Ras Conformational Ensembles, Allostery, and ... - ACS Publications

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Ras Conformational Ensembles, Allostery, and Signaling Shaoyong Lu,†,‡ Hyunbum Jang,‡ Serena Muratcioglu,§ Attila Gursoy,∥ Ozlem Keskin,§ Ruth Nussinov,*,‡,⊥ and Jian Zhang*,† †

Department of Pathophysiology, Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai, 200025, China ‡ Cancer and Inflammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, National Cancer Institute, Frederick, Maryland 21702, United States § Department of Chemical and Biological Engineering and ∥Department of Computer Engineering, Koç University, Rumelifeneri Yolu, 34450 Sariyer Istanbul, Turkey ⊥ Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Sackler Institute of Molecular Medicine, Tel Aviv University, Tel Aviv 69978, Israel ABSTRACT: Ras proteins are classical members of small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and inactivation is terminated by GTPase-activating proteins that accelerate the intrinsic GTP hydrolysis rate by orders of magnitude. In this review, we focus on data that have accumulated over the past few years pertaining to the conformational ensembles and the allosteric regulation of Ras proteins and their interpretation from our conformational landscape standpoint. The Ras ensemble embodies all states, including the ligand-bound conformations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, transition states, and nonfunctional states serving as a reservoir for emerging functions. The ensemble is shifted by distinct mutational events, cofactors, post-translational modifications, and different membrane compositions. A better understanding of Ras biology can contribute to therapeutic strategies.

CONTENTS 1. Introduction 2. Sequences of Ras 3. Structures of Ras 3.1. X-ray Crystal Structures of Ras Catalytic Domain 3.2. NMR Structures of Ras Catalytic Domain 3.3. Models of Full-Length Ras 3.4. Identified Driver and Anchor Atoms for Ras 4. Differential Dynamics of the Catalytic Domains of Ras Isoforms 5. Conformational Path from the Active Ras−GTP to the Inactive Ras−GDP 5.1. Identifying the Conformational Path by Computational Methods 5.2. Capturing Intermediates in the Conformational Path by Crystallography 5.2.1. A59G H-Ras 5.2.2. Q61G H-Ras 6. Conformational Ensemble of Ras−GTP in Solution 6.1. Ras−GTP Exists in Active and Inactive States 6.2. Oncogenic Mutations Shift the Ras−GTP Ensemble toward the Active State

© 2016 American Chemical Society

6.3. Anti-Ras Intrabody Inhibits the Active State of Ras−GTP 6.4. Compounds Stabilize the Inactive State of Ras−GTP 6.4.1. Kobe0065 Family Compounds 6.4.2. Zn2+/Cu2+−Cyclen Complexes 6.4.3. Zn2+−BPA Complexes 7. Stabilization of Ras−GDP by S-IIP Binding Compounds 8. Allosteric Switch of Ras−GTP to the On State 9. Conformational Equilibrium between the On and Off States of Ras−GTP 10. Regulation of Ras 10.1. Activation of Ras by SOS 10.2. Inactivation of Ras by GAP 11. Association of Ras−GTP with Effector Proteins 11. 1. Interaction with Raf 11.2. Interaction with PI3K 11.2.1. Calmodulin’s Full Activation of PI3Kα in the Case of Oncogenic K-Ras4B 11.3. Interaction with RalGDS

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Special Issue: Protein Ensembles and Allostery

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Received: September 15, 2015 Published: January 27, 2016 6607

DOI: 10.1021/acs.chemrev.5b00542 Chem. Rev. 2016, 116, 6607−6665

Chemical Reviews 11.4. Interaction with Bry2 11.5. Interaction with PLCε 11.6. Interaction with NORE1A 11.7. Interaction with Grb14 12. RAS Dimers and Higher Architectural States 13. RAS Interaction with the Membrane 13.1. Interactions of the HVR Peptide with Membranes 13.2. K-Ras4B Membrane Localizations 13.3. Interactions of K-Ras4B with Membranes 14. Conclusions Author Information Corresponding Authors Notes Biographies Acknowledgments References

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1. INTRODUCTION Ras genes were first identified and characterized as transduced oncogenes in the Harvey and Kirsten strains of acutely transforming retroviruses.1 In 1982, Chang et al.2 found mutationally activated Ras genes in human cancers. This pioneering study prompted further analysis of Ras genes and their products. Decades of research have revealed that Ras regulates signaling pathways essential for the growth, proliferation, and differentiation of cells.3−8 Epidermal growth factor (EGF) binding to the extracellular domain of receptor tyrosine kinases (RTKs)9 such as epidermal growth factor receptor (EGFR) results in dimerization and activation of EGFR. The activated EGFR recruits Ras-specific guanine nucleotide exchange factors (GEFs) [e.g., SOS (son of sevenless)] via the adaptor proteins SHC (SHC-transforming protein) and Grb2 (growth factor receptor-bound protein 2). SOS exchanges GDP by GTP and activates Ras. Active Ras− GTP dimerizes and binds to its downstream effector, Raf kinase, thereby leading to dimerization and activation of Raf.10,11 In the Ras−Raf−MEK−ERK pathway12,13 (Figure 1), active Raf dimer phosphorylates and activates MEK1/2 (extracellular signal regulated kinase 1/2), which in turn phosphorylates and activates ERK1/2. Active ERK1/2 phosphorylates and activates ELK-1 (E26 oncogene homology 1-like gene 1), a transcription factor. ELK-1 binds to its cofactor, SRF (serum response factor) dimer, leading to transcription activation and cell proliferation.14 In the Ras− PI3K−Akt pathway15−17 (Figure 1), active Ras−GTP recruits PI3K (phosphatidylinositol 3-kinase) to the plasma membrane (PM), where its substrate PIP2 (phosphatidylinositol-3,4bisphosphate) is located. PI3K phosphorylates PIP2 to produce PIP3 (phosphatidylinositol-3,4,5-bisphosphate), a process that can be reversed by PTEN (phosphatase and tensin homologue). PIP3 recruits both Akt and PDK1 (3phosphoinositide-dependent kinase 1) to the PM, where PDK1 phosphorylates and activates Akt. Active Akt phosphorylates the mTOR (mammalian target of rapamycin) complex, which regulates cell survival and proliferation. Ras proteins are quintessential members of small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states.18−26 Activation is tightly regulated by GEFs, which catalyze the exchange of GDP by GTP. Active Ras−GTP can bind and activate downstream effectors, including Raf kinase, PI3K, and

Figure 1. Ras signaling in the mitogen-activated protein kinase (MAPK), Raf−MEK−ERK pathway, and phosphatidylinostiol 3-kinase (PI3K)/Akt pathway.

Ral guanine nucleotide dissociation stimulator (RalGDS).27−29 Ras inactivation is mediated by GTPase-activating proteins (GAPs), which accelerate the intrinsic GTP hydrolysis rate of Ras by several orders of magnitude.30 Ras mutations that impair GTPase activity are insensitive to GAPs, rendering mutant Ras proteins persistent in their active GTP-bound state, thereby prolonging the downstream signaling associated with oncogenic cell growth.31−35 Oncogenic mutations in Ras are found in approximately 30% of human cancers, with the mutations frequently found in lung, colon, and pancreatic cancers.10,36−42 Despite the more than 3 decades of efforts, no effective inhibitors of Ras oncoproteins have been successful in the clinic, rendering Ras proteins still “undruggable”.43 This is attributed to their relatively smooth surface with few pockets where a molecule might bind tightly. Although it represents a key challenge for taming Ras, the importance of Ras signaling pathways in cancer therapy has not gone unnoticed by the scientific and industrial community.44−54 For example, in 2013, the US National Cancer Institute launched the Ras Initiative, which unites academia and the pharmaceutical and biotech industries to find a viable approach to tackle Ras-driven cancers.55 In recent years, studies of Ras have witnessed a renaissance fueled by unraveling the conformational dynamics and identifying new pockets in Ras.56−63 For instance, based on X-ray crystallography and NMR spectroscopy, two conformational states, inactive state 1 and active state 2, were observed in Ras−GTP. Ras exists in a conformational equilibrium between the inactive and active states, and the transition from the inactive state to the active state is mediated by an allosteric switch mechanism.64−69 Small-molecule Kobe0065-family compounds and cyclen−metal complexes bind specifically to the inactive state, which shift the conformational ensemble of Ras to the inactive state,70 thereby blocking the signaling of oncogenic Ras. Small-molecule compounds SML-8-73-171,72 and SML-10-70-1,71 acting as active site inhibitors of oncogenic G12C K-Ras mutant, provide a feasible therapeutic strategy for 6608


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inhibition of Ras signaling. Significantly, Ostrem et al.73 identified a new allosteric regulatory site on oncogenic G12C K-Ras mutant using a disulfide-fragment-based screening approach. In this review, we focus on data related to the conformational ensemble and allosteric regulation of Ras proteins that have accumulated over the past few years using X-ray crystallography, NMR spectroscopy, biochemical studies, computer simulations, and their interpretation from our conformational landscape standpoint. This view is the distinctive feature differentiating the many excellent cell biology reviews of the Ras protein from the current one. The deeper mechanistic insight on the molecular level that this allows embodies the essence of the “second molecular biological revolution”.74 The structure−function paradigm fomented by this revolution posited that all forms of life conform to the laws of quantum mechanics and structural chemistry; it further led to the powerful idea of the energy landscape75 and the recognition that biomolecules are dynamical objects that are always interconverting between structures with varying energies, inspiring the idea of the dynamical statistical description of the molecules.76−79 This revolution allows one to ask how and why one-dimensionally connected biomolecules not only organize themselves in three dimensions, as we see in crystal structures, but function and why and how changes in the linear connection, i.e., mutations, can lead to dysfunction.80 Similarly, it allows one to ask how a drug designed to block the inactive state of the proteinas we shall see below for GDP-bound Rasworks to block its active GTP-bound signaling state.81 The free energy landscape and a dynamical statistical description of macromolecules afford an insight into how changes in the intra- and intermolecular interactionseven if seemingly slightcan result in an alternative pathway taking over or in constitutive activation. It speaks the language of relative conformational stabilities, transitions among conformational states, dynamic conformational distributions, and population shifts.82−89 Figure 2 depicts a schematic free energy landscape diagram, illustrating the pre-existence of substates, including mutational states, complexed states, and druginteracting states. There is a large number of inactive protein substates; there is, however, only a single active state where all chemical groups participating in a reaction are accurately positioned with respect to each other. Below, as a background, we first outline the sequences and structures of Ras proteins, followed by a description of the inactive and active states, the conformational transition of the active to the inactive states, the effects of oncogenic mutations on Ras conformers and the ensemble, allosteric regulation of Ras, interactions of Ras with effectors, Ras dimers, and the interactions of Ras with the membrane, which are expected to contribute to the discovery and development of small-molecule Ras inhibitors employing protein-structure-guided design approaches. These are all put in the framework of Ras conformational ensembles. Other proteins can be usefully described in a similar vein.

Figure 2. Schematic description of the shift in the free energy landscape following mutational states, complexed states, and druginteracting states. (A) Middle: The conformational distribution shifts from the favorable active conformation of Ras (top) to the inactive conformation of Ras (bottom). Left: Conformational change of Ras due to GAP-mediated or intrinsic GTP hydrolysis highlighted by superimposing the two Ras structures with GppNHp-bound H-Ras (PDB ID 5P21) (switch I and switch II regions, red) and GDP-bound H-Ras (PDB ID 4Q21) (switch I and switch II regions, green). Right: Conformational change of Ras due to binding of the small-molecule inhibitor Kobe2601 highlighted by superimposing the two Ras structures with GppNHp-bound H-Ras (PDB ID 5P21) (red) and GppNHp-bound H-Ras in complex with Kobe2601 (PDB ID 2LWI) (green). (B) Middle: The relative distribution favors more of the active conformation in the ensemble. Left: Conformational change of Ras due to oncogenic mutations highlighted by superimposing the two Ras structures with wild-type GppNHp-bound H-Ras (PDB ID 5P21) (red) and G12D GppNHp-bound H-Ras (PDB ID 1AGP) (green). Right: Conformational change of Ras due to the binding of downstream effectors, RafRBD (pink), highlighted by superimposing the two Ras structures with GppNHp-bound H-Ras (PDB ID 5P21) (red) and GppNHp-bound H-Ras in complex with RafRBD (PDB ID 4G0N) (green). (C) Right: The conformational distribution shifts from the favorable inactive conformation of Ras (top) back to the active conformation of Ras (bottom). Left: Conformational change of Ras due to SOS-catalyzed nucleotide exchange highlighted by superimposing the two Ras structures with GDP-bound H-Ras (PDB ID 4Q21) (red) and GppNHp-bound H-Ras (PDB ID 5P21) (green). (D) Right: The relative distribution favors more of the inactive conformation in the ensemble. Left: Conformational change of Ras due to binding of a covalent inhibitor, compound 6, attached to C12 highlighted by superimposing the two Ras structures with G12C GDP-bound K-Ras4B (PDB ID 4L9S) (red) and G12C GDP-bound K-Ras4B in complex with compound 6 (PDB ID 4LUC) (green).

2. SEQUENCES OF RAS So far, three Ras genes have been identified in the mammalian genome. These encode multiple isoforms, including H-Ras, KRas, and N-Ras.90,91 K-Ras can be found as two splice variants designated K-Ras4A and K-Ras4B. Both are oncogenic when KRas is mutated, but the mechanisms of K-Ras4A and K-Ras4B membrane trafficking appear distinct, suggesting that the 6609


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Figure 3. Multiple sequence alignment of the amino acids in H-Ras, N-Ras, K-Ras4A, and K-Ras4B. In the sequence, hydrophobic, polar/glycine, positively charged, and negatively charged residues are colored black, green, blue, and red, respectively. The nonidentity of residues in the alignment is indicated by underlined texts. In the hypervariable region (HVR) sequences, the purple boxes denote the palmiltoylated cysteines and orange boxes indicate the farnesylated cysteines. A distinguishing feature of the HVR of K-Ras4B is the presence of a polybasic stretch. Reproduced with permission from ref 332. Copyright 2015 Informa Healthcare.

G13 (14%), respectively. Each mutation in each isoform occupies a distinct conformational state. Currently, the molecular mechanisms corresponding to the varying incidences of Ras mutations in different tumor types and specific associations of each Ras isoform with particular cancers remain unresolved.106 The C-terminal 22 or 23 amino acids of the HVR are probably responsible for the biological differences across Ras isoforms because this is the only region that differs markedly in sequence among them (Figure 3). As such it presents distinct preferred conformational states and interactions and may provide clues for isoform-specific differences in network signaling and oncogenic potential.107 The C-terminal protein sequences conserved in the HVR CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid),108−110 are necessary for Ras recruitment to the inner PM for normal biological function.111−113 Mutation of the CAAX motif in the HVR has been demonstrated to demolish PM localization and Ras signaling.114 The initial triplet of modifications of the CAAX motif is a prerequisite for all Ras proteins, encompassing farnesylation at cysteine within the CAAX motif, AAX proteolysis, and carboxyl methylation of the resulting Cterminal prenylcysteine.115,116 In order to insert the HVR into the PM, Ras proteins require a second signal. The HVR of KRas4B contains multiple lysines (K175−K180) that serve as a polybasic stretch, which is capable of associating with the negatively charged phospholipids in the inner PM leaflet of acidic membranes through electrostatic interactions. Owing to a paucity of the polybasic stretch in the HVR of H-/N-Ras and to a lesser extent K-Ras4A for association with negatively charged membranes, all three are additionally palmitoylated on cysteine residues (C181 and C184 for H-Ras, C181 for N-Ras, and C180 for K-Ras4A) within the HVR, immediately proximal to the CAAX motif.117,118 Palmitoylation strengthens the lipophilicity of the HVR and enables association with a zwitterionic PM. Its linkage to the HVR is reversible. In addition to post-translational modifications (PTMs) on the HVR, K-Ras4B has been reported to be acetylated on K104 at the catalytic domain.119 Molecular dynamics (MD) simulations of nonacetylated and acetylated K-Ras4B G12V mutant suggested that the K104 acetylation affects the conformational

development of anti-K-Ras drugs that interfere with membrane trafficking should consider the different modes of membrane targeting of the two K-Ras splice variants and their states.92,93 H-Ras, K-Ras4A, and N-Ras have 189 amino acids, while KRas4B has 188 amino acids. Multiple sequence alignment (Figure 3) reveals that the total sequence identity between the four isoforms is approximately 79%. Remarkably, the catalytic domain (residues 1−166) of the four isoforms shares highly conserved sequence identity (∼89%), while extremely low sequence identify (∼8%) is observed in the hypervariable region (HVR) of the four isoforms (residues 167−189 for HRas, K-Ras4A, and N-Ras; 167−188 for K-Ras4B).94−96 Despite a high degree of sequence identity across Ras isoforms, the frequency and distribution of Ras mutations are not equivalent. The Catalog of Somatic Mutations in Cancer (COSMIC)97 confirms that K-Ras is the most frequently mutated isoform in Ras-driven cancers (86%), followed by N-Ras (11%) and H-Ras (3%).98 Mutated isoforms tend to associate with particular tumor types:6,24,29,55−57 K-Ras mutations occur at very high frequency in pancreatic ductal adenocarcinoma and lung and colon tumors; N-Ras mutations are commonly detected in hematopoietic tumors and in malignant melanomas; H-Ras mutations are the most frequent in bladder tumors and head and neck squamous cell carcinoma. Significantly, 98% of the oncogenic Ras mutations are found at amino acid residues G12, G13, and Q61, the mutations of which impair intrinsic and GAP-mediated GTP hydrolysis, locking mutant Ras in the active Ras−GTP state, although additional mutations at residues L19, Q22, E37, K117, A146, and R164 have been reported in colorectal tumors and chronic myelomonocytic leukemia.99−102 Nevertheless, cancer-associated Ras isoforms exhibit an intimate link to residue substitutions.4,37,64,103−105 For example, K-Ras G12 mutations (89%) are predominant in human cancers, followed by G13 (9%) and Q61 (1%) mutations (G12 > G13 > Q61). Moreover, the G12D mutation is arguably the most prevalent mutation among the three frequent G12C (14%), G12D (36%), and G12V (23%) mutations. In addition, G13D (7%) and Q61H (0.6%) mutations are also observed. Unlike K-Ras, mutational frequencies in H-Ras and N-Ras follow the order G12 (55%) > Q61 (36%) > G13 (8%) and Q61 (60%) > G12 (25%) > 6610


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Table 1. Representative Structures of Ras Proteins isoforms

methods

PDB ID

H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras H-Ras K-Ras4B K-Ras4B K-Ras4B K-Ras4B K-Ras4B K-Ras4B N-Ras

X-ray X-ray NMR NMR X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray

5P21 4Q21 2LWI 1CRP 1WQ1 1HE8 3KUD 4G0N 1NVV 1XD2 1LFD 3DDC 2C5L 4K81 1K8R 3K8Y 3L8Y 2UZI 3RS0 4DLR 3GFT 4LPK 4M1Y 4EPV 4DSU 4Q03 3CON

resolution (Ǻ ) 1.35 2.00

2.50 3.00 2.15 2.45 2.18 2.70 2.10 1.80 1.90 2.40 3.00 1.30 2.02 2.00 1.40 1.32 2.27 1.50 1.49 1.35 1.70 1.20 1.65

nucleotide GppNHp GDP GppNHp GDP GDP/AlF3 GppNHp GDP GppNHp GTP GDP GppNHp GppNHp GTP GTP GppNHp GppNHp GppNHp GTP GppNHp GppNHp GppNHp GDP GDP GDP GDP GDP GDP

effectors

p120GAP PI3K RafRBD RafRBD SOS SOS RalGDS NORE1A PLC Grb14 Bry2RBD

ligands

ACT/Ca2+/DTU

ACT/Ca2+ cyclen/Zn2+/Ca2+ anti-Ras FV Ca2+ DTU/Ca2+

21S 0QX BZI 2XE

refs 145 146 70 189 272 163 314 162 249 255 164 165 166 167 168 175 235 227 173 169 / 73 73 261 252 59

preferences of Ras isoforms.33,34 Apart from the engagement of the PM, which has been well-established as a platform for Ras to signal,131,132 a host of studies using imaging technology such as fluorescence resonance energy transfer (FRET) and quantitative fluorescent microscopy in living cells have pinpointed that interactions of Ras proteins with endomembranes (i.e., the endoplasmic reticulum, Golgi, and vesicular compartments) serve as other platforms for Ras signaling,133,134 and numerous excellent reviews have addressed this notion.135−142

stability of the switch II region to obstruct GEF-induced nucleotide exchange. This hypothesis is supported by an acetylation mimetic mutation (K104Q) in K-Ras4B, which demonstrates that K104 acetylation suppresses GEF-induced nucleotide exchange and inhibits in vitro K-Ras4B transforming activity, indicating that K104 acetylation is a negative regulatory modification on K-Ras4B.119 In addition, K104 and K147 ubiquitination of K-Ras4B, T35 glycosylation, R41 and R128 ADP-ribosylation, and C118 nitrosylation and glutathionylation of H-Ras at the catalytic domain have also been observed to influence Ras trafficking and signaling.120−127 The C185 farnesylation allows K-Ras4B to insert the prenyl group in the HVR into a disordered PM.128 Unlike palmitoylation, farnesylation is irreversible. Recently, we performed MD simulations of the HVR peptide with the farnesyl at C185 in two types of zwitterionic lipid bilayers, containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and anionic lipid bilayers containing DOPC/1,2dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) (mole ratio 4:1). 129 The results showed that the farnesyl group spontaneously inserts into the disordered lipid microdomains, while the rigid microdomains restrict the farnesyl group penetration. Further simulations of an additional phosphorylation at S181 showed that S181 phosphorylation prohibits spontaneous farnesyl membrane insertion. These data indicate that the HVR PTMs in K-Ras4B play a pivotal role in targeting microdomains of the PM, suggesting an additional function for HVR in regulation of Ras signaling. Unlike K-Ras4B, GDPbound H-Ras associates initially with rigid lipid rafts; subsequently, it shifts to the disordered PM after GTP loading.130 These differences reflect the different membrane

3. STRUCTURES OF RAS H-Ras, as a prototype in studies of Ras biology, was the first structure, determined in 1988.143 The structure, obtained by Xray crystallography, solved the H-Ras catalytic domain in complex with a GDP, which revealed that the overall topology of the catalytic domain is a globular fold. The crystal structure of H-Ras catalytic domain in complex with a nonhydrolyzable GTP analog [guanosine-5′-(β,γ-imido) triphosphate, GppNHp] was determined in 1990.144−146 Comprehensive information on the structure and function of Ras was gained in the following year from the determination of crystal structures of H-Ras/K-Ras4B complexed with GTP, GDP, GppNHp, and double-modified nonhydrolyzable GTP analogues DABP− GppNHp and PDA−GppNHp;147 various oncogenic and nononcogenic mutants of H-Ras/K-Ras4B, such as G12C,73 G12D,148 G12V,144,149,150 G12P,148 G12R,144 G13D,151 S17N,152 T35S,153 D38E,144 T50I,154 A59G,155 A59T,156 G60A,157 Q61H,144 Q61I,158 Q61K,158 Q61L,144 Q61 V,158 K117R,159 Y137E,160 Y137F,160 and K147A161 mutations; and H-Ras complexed with downstream effectors, including Raf (PDB ID 4G0N),162 PI3K (PDB ID 1HE8),163 RalGDS (PDB 6611


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Figure 4. (A) Cartoon representation of the crystal structure of GppNHp-bound H-Ras (PDB ID 5P21). The helices, strands, and loops are colored red, yellow, and gray, respectively. The P-loop, switch I, and switch II regions are colored lime, pink, and blue, respectively. GppNHp and Mg2+ are depicted by stick models and a green sphere, respectively. (B) Cartoon representation of the crystal structure of GDP-bound H-Ras (PDB ID 4Q21). (C) The positions of the three most frequently oncogenic mutated residues, G12, G13, and Q61, in the GppNHp-bound H-Ras. (D) Backbone superimposition of crystal structure of GppNHp-bound H-Ras (pink) onto that of GDP-bound H-Ras (cyan). (E) The coordination modes of Mg2+ in the GppNHp-bound H-Ras. (F) Surface representation of GppNHp-bound H-Ras. (G) The coordination modes of Mg2+ in the GDP-bound H-Ras. (H) Surface representation of GDP-bound H-Ras. (I) The different orientation of Y32 between GppNHp- (pink) and GDPbound H-Ras (cyan). Y32 is depicted by stick models. Reproduced with permission from ref 54. Copyright 2015 The Wiley Press.

ID 1LFD),164 NORE1A (PDB ID 3DDC),165 phospholipase C (PDB ID 2C5L),166 adaptor protein Grb14 (PDB ID 4K81),167 and Byr2 kinase (PDB ID 1K8R).168 Subsequently, the NMR structures of H-Ras catalytic domain in complex with GDP (PDB ID 1CRP) and GppNHp (PDB ID 2LCF and 2LWI) were determined, revealing the solution structure and dynamics of Ras proteins. The high-resolution structures of GppNHpbound H-Ras catalytic domain complexed with calcium acetate, dithioerythritol, and dithiothreitol provide insights into the existence of conformational equilibrium between “on” and “off” states in solution.169 Room-temperature X-ray crystallography has uncovered a previously hidden structural ensemble of GppNHp-bound H-Ras in protein crystals.170 Recently reported crystal structures of Ras in complex with noncovalent or covalent inhibitors have advanced the understanding of the mechanisms of Ras inhibition.72,171 To date, only one crystal structure of N-Ras in complex with GDP has been reported (PDB ID 3CON) and structures of K-Ras4A have not yet been determined. Of note, no structures of full-length Ras are currently available; Ras structures are determined exclusively with a truncated catalytic domain. When determination of the full-length Ras structure was attempted, no HVR electron density was obtained. Representative structures of Ras proteins and the corresponding PDB ID are listed in Table 1.

3.1. X-ray Crystal Structures of Ras Catalytic Domain

The X-ray crystal structures of H-Ras catalytic domain complexed with GppNHp (PDB ID 5P21)145 and GDP (PDB ID 4Q21)146 revealed that the catalytic domain of Ras is composed of the quintessential G-domain structure containing the catalytic machinery with 6 β-strands (β1−β6) flanked by 5 α-helices (α1-α5) and 10 connecting loops (Figure 4A,B). The functional P-loop (residues 10−17), switch I (residues 30−38), and switch II (residues 59−76) regions constitute the active site for GTP/GDP and interaction sites for effector proteins, including Raf, PI3K, RalGDS, and GAP.172 These structural elements are within the first “effector lobe” (residues 1−86) half of the catalytic domain, which has 100% sequence identity across Ras isoforms.173,174 The remainder of the molecule, containing a more variable sequence among Ras isoforms (Figure 3), serves as the second “allosteric lobe” (residues 87−166) half of the catalytic domain, which has an allosteric site.175 The allosteric lobe has recently gained increasing recognition as a strategy for designing more selective drugs for Ras isoforms, because in vitro studies have elucidated isoform-specific PM microlocalization patterns.176 However, an atomic resolution view of the Ras−membrane association is still unavailable. 6612


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Figure 5. (A) Cartoon representation of the crystal structure of GppNHp-bound K-Ras4B (PDB ID 3GFT). (B) Cartoon representation of the crystal structure of GDP-bound K-Ras4B (PDB ID 4LPK). (C) Backbone superimposition of the crystal structure of GppNHp-bound K-Ras4B (pink) onto that of GDP-bound K-Ras4B (cyan).

and GDP (PDB ID 4LPK) are similar to the corresponding structures of GppNHp- and GDP-bound H-Ras (Figure 5A,B). It also contains 6 β-strands, 5 α-helices, and 10 connecting loops. Backbone superimposition of the crystal structure of GppNHp-bound K-Ras4B onto that of GppNHp-bound H-Ras yields a backbone RMSD of 0.96 Å, and the RMSD between GDP-bound K-Ras4B and H-Ras is 0.97 Å for the backbone atoms. Backbone superimposition of GppNHp- and GDPbound K-Ras4B yields a backbone RMSD of 1.91 Å and also reveals that the switch I and switch II regions exhibit the most significant conformational differences between GppNHp- and GDP-bound K-Ras4B (the backbone RMSD is 0.58 Å, if the switch I and switch II regions are excluded in the calculation) (Figure 5C), which is in good agreement with the structures of GppNHp- and GDP-bound H-Ras. Importantly, the paradigms of nucleotide−Ras interactions, Mg2+ coordination modes, and arrangement of critical residues in the active site of GppNHp (GDP)-bound K-Ras4B are highly similar to those of GppNHp (GDP)-bound H-Ras. The crystal structure of N-Ras catalytic domain in complex with GDP has been reported (PDB ID 3CON). However, the electron density for residues in the switch II region is not welldefined, as evidenced by their high-temperature factors. By excluding the switch II region from the analysis, the backbone RMSD between GDP-bound H-Ras and N-Ras is 0.68 and 0.49 Å between GDP-bound K-Ras4B and N-Ras, suggesting subtle conformational differences between GDP-bound H-Ras, KRas4B, and N-Ras. Overall, the interaction modes between GDP and the protein in N-Ras are similar to those of H-Ras and K-Ras4B. Two N-Ras mutations in the catalytic domain, T50I and G60E, exhibit typical clinical features of patients with Noonan syndrome.154 T50, residing in the β2−β3 loop connecting the switch I and switch II regions, is exposed to solvent and is not directly involved in contacts with GTP or GDP. Biochemical characterization showed that the G60E mutant, like the G12V mutant, is resistant to intrinsic and GAPmediated GTP hydrolysis, rendering the N-Ras G60E mutant constitutive in the active, GTP-bound state. This can be explained: the backbone nitrogen atom of G60 in the switch II region forms a hydrogen bond with the γ-phosphate of GTP, and the mutation of Gly to Glu would cause electrostatic repulsion between E60 and the γ-phosphate, thereby resulting in impairment of intrinsic and GAP-mediated GTP hydrolysis. However, the T50I mutation does not markedly affect either the intrinsic ormore importantlythe GAP-mediated GTP hydrolysis. MD simulations of a wild-type Ras-membrane lipid complex showed that the hydroxyl group of T50 directly

Two of the three most frequent oncogenic mutations, G12 and G13, are located in the P-loop, while Q61 is located in the switch II region (Figure 4C). Arguably the most remarkable differences in the GppNHp- and GDP-bound H-Ras are in the switch I and switch II regions (Figure 4D), revealing a crucial role of the nucleotide-mediated cooperativity between the two switch regions in the conformational transition.177 The rootmean-square deviation (RMSD) between the whole catalytic domains of GppNHp- and GDP-bound H-Ras is 1.58 Å for the backbone heavy atoms. If the switch I and switch II regions are excluded from the calculation, the RMSD is only 0.66 Å. In the GppNHp-bound H-Ras, the γ-phosphate of GppNHp forms two hydrogen bonds with the residue G60 in the switch II region and residue T35 in the switch I region. The Mg2+ in the active site is bidentately coordinated by the nonbridging oxygen atoms of the β- and γ-phosphates, T35 in the switch I region, S17 in the P-loop, and two water molecules (Figure 4E).178−180 Quantum mechanics/molecular mechanics (QM/MM) simulations of GTP-bound H-Ras/Mg2+ complex illustrated that Mg2+ provides a temporary storage for electrons taken from the triphosphate and that after bond cleavage and γ-phosphate release it returns them back to the diphosphate, thereby contributing to catalysis.181,182 Overall, the arrangement of the three functional P-loop, switch I, and switch II regions forms the closed conformation of the GTP-binding site (Figure 4F), which contributes to intrinsic GTP hydrolysis. Following GTP → GDP hydrolysis, the β-phosphate of GDP has no direct interactions with residues T35 and G60; coupled with the loss of coordination interaction between Mg2+ and T35 (Figure 4G), these lead to extensive remodeling of the switch I and switch II regions. As a result, the nucleotide-binding site in the GDP-bound H-Ras is in the open conformation (Figure 4H), which allows GDP dissociation from the nucleotide-binding site. Another distinguishing feature is the difference in the sidechain conformation of switch I residue Y32 in the GppNHpand GDP-bound H-Ras. In the GppNHp-bound state, Y32 is in the “up” conformation that points to the solvent, whereas in the GDP-bound state it undergoes a large flip and shifts to the interaction site where it resides in the “down” conformation (Figure 4I). Furthermore, the outward displacement of the switch II region after GTP hydrolysis results in the catalytic residue Q61 pointing away from the active site. An impressive body of biochemical experiments and computational studies has revealed that Q61 plays a critical role in both intrinsic and GAP-mediated GTP hydrolysis.183−186 The overall three-dimensional (3D) structures of K-Ras4B catalytic domain complexed with GppNHp (PDB ID 3GFT) 6613


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Figure 6. (A) Cartoon representation of the backbone superimposition of the first 20 lowest-energy NMR structures of GDP-bound H-Ras (PDB ID 1CRP). The switch I and switch II regions are colored pink and blue, respectively. The GDP in the first respective structure is shown as stick models. (B) Backbone superimposition of the second 20 lowest-energy NMR structures of GDP-bound H-Ras (PDB ID 1CRR). (C) Backbone superimposition of the average of 40 NMR structures (PDB ID 1CRQ, pink) onto the crystal structure of GDP-bound H-Ras (PDB ID 4Q21, cyan).

Figure 7. (A) Cartoon representation of the backbone superimposition of the 20 lowest-energy NMR structures of GppNHp-bound H-RasT35S (PDB ID 2LCF). The P-loop, switch I, and switch II regions are colored lime, pink, and blue, respectively. The GppNHp in the first structure is shown as stick models. (B) Comparison of the NMR and the crystal structures in the switch I region of H-Ras. The NMR structures of H-RasT35S− GppNHp (pink), the crystal structures of H-Ras−GppNHp (PDB ID 5P21, blue), and H-Ras−GDP (PDB ID 4Q21, green), and the nucleotide-free form of H-Ras (PDB ID 1BKD, cyan) are shown.

interacts with the polar heads of membrane phospholipids,154 which stabilizes the Ras orientation with respect to the membrane. Thus, it is anticipated to decrease downstream signaling, presumably by keeping N-Ras in a less productive signaling conformation.187 In contrast, the T50I mutation disrupts the Ras−membrane interactions and subsequently alters Ras orientation with respect to the membrane. This strengthens the interaction of GTP-bound Ras with its downstream effectors, thereby leading to enhanced downstream signaling, presumably retaining N-Ras in a more productive signaling conformation.187

to probe the conformational dynamics of proteins in solution, which can provide detailed dynamic properties of proteins that are mostly lost in crystal structures where a preferred conformation under the crystallization conditions is frozen out because of crystal packing forces. Using heteronuclear 3D and 4D NMR spectroscopy, a highresolution solution structure of H-Ras catalytic domain in complex with GDP has been determined.189 A total of 40 final structures were computed from the experimental restraints. As shown in Figure 6A,B, the topology of the fold in the H-Ras solution structure (PDB ID 1CRP and 1CRR)189 is identical to that observed in the X-ray diffraction studies. The secondary structure elements, described as α/β structure with five αhelices and six β-strands, are also the same as those published structures determined by X-ray crystallography. The solution NMR structures of GDP-bound H-Ras reveal that the segments comprising the switch II region, and to a lesser extent the switch I region, are flexible in solution. The pronounced flexibility of the switch II region observed in the NMR studies suggests the existence of multiple conformations of the switch II region in solution, which provides an unprecedented insight into the activation of the GTPase hydrolysis by GAP. When GAP binds to Ras, the mobility of the switch II region is restricted and the catalytically active conformation becomes

3.2. NMR Structures of Ras Catalytic Domain

The determination of high-resolution X-ray crystal structures of Ras proteins complexed with GDP and GppNHp demonstrates major conformational changes in the switch I and switch II regions.143,145,146 GTP binding to Ras enables the switch regions to orient favorably for Ras to associate with its effectors, such as Raf and PI3K. However, static crystal structures may be inadequate to uncover structural dynamics information about Ras proteins and how they interact with their effectors, because in the absence of crystal packing, the protein has significant structural fluctuations in solution.188 Nuclear magnetic resonance (NMR) spectroscopy is the most suitable technique 6614


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Figure 8. (A) Cartoon representation of full-length K-Ras4B−GTP. The catalytic domain and the HVR are colored gray and orange, respectively. (B) Surface representation of full-length K-Ras4B−GTP. (C) Residues K178, K180, K182, and K184 of the HVR form electrostatic interactions with the residues E37, D38, D33, and E31 of the switch I region in the full-length K-Ras4B−GTP. The salt bridges are depicted by blue dotted lines. (D) Cartoon representation of full-length K-Ras4B−GDP. The HVR runs between the L2 loop and β2 strand. (E) Surface representation of full-length K-Ras4B−GDP. (F) Residues K176, K178, K180, and K182 of the HVR form electrostatic interactions with the residues E37 and D38 of the switch I and E62 of the switch II region in the full-length K-Ras4B−GDP. (G) Backbone superimposition of full-length K-Ras4B−GDP (pink) onto the crystal structure of H-Ras−GppNHp catalytic domain (cyan) in complex with RafRBD (light blue) (PDB ID 4G0N).

contrast to the moderate and subtle structural changes of the switch II region and the P-loop, respectively (Figure 7A). This is pronouncedly different from the solution structure of GDPbound H-Ras (Figure 6), where the switch II region exhibits a wide range of conformational changes with moderate restraint of the switch I region. The backbone structures of the switch I region of GppNHp-bound H-RasT35S solution structure are further superimposed on those of the X-ray structures of GppNHp- (PDB ID 5P21) and GDP-bound (PDB ID 4Q21) H-Ras and the nucleotide-free form of H-Ras in complex with SOS (PDB ID 1BKD).193 The results show that some of the 20 switch I structures resemble those of the nucleotide-free H-Ras in complex with SOS and the crystal structures of GppNHpand GDP-bound H-Ras (Figure 7B), indicating that in solution the switch I region of GppNHp-bound H-RasT35S bears a wide range of structural variability.

stabilized, resulting in a rapid switching off of the signal. In the absence of GAP, the population of the catalytically active conformation in solution is much lower owing to the mobility of the switch II region. In this scenario, intrinsic GTP hydrolysis is markedly slower compared to GAP-mediated GTP hydrolysis.190 Backbone superimposition of the average of the 40 solution structures (PDB ID 1CRQ) and the X-ray crystal structure of GDP-bound H-Ras (PDB ID 4Q21) shows that the two structures are very similar, except for the switch I and switch II regions (Figure 6C), which indicates that NMR structures are mobile. The RMSD between the two structures is 1.04 Å for the backbone atoms if both switch regions are excluded from the calculation. Attempts to determine the solution structures of wild-type GppNHp-bound Ras using multidimensional heteronuclear NMR spectroscopy have been frustrating, since chemical exchange processes at intermediate rates on the NMR time scale cause broadening for the majority of the residues in the Ploop and switch I and switch II regions.191 However, solution structures of T35S H-Ras mutant in complex with GppNHp have recently been determined (PDB ID 2LCF).192 This mutation is able to eradicate the slow conformational exchange process.191 The topology of GppNHp-bound H-RasT35S solution structure is consistent with the canonical nucleotidebinding Ras proteins. Comparison of the 20 NMR structures reveals that the switch I region displays a marked fluctuation, in

3.3. Models of Full-Length Ras

Currently, no crystal or NMR structures of full-length Ras are solved, leading to the lack of a detailed, atomic-resolution understanding of the association of the HVR with the catalytic domain. Previously, Thapar et al.,194 using NMR spectroscopy, pointed out that the HVR of H-Ras transiently interacts with the catalytic domain. To further uncover the interaction site of HVR with the catalytic domain, NMR spectroscopy was performed to compare the properties of truncated K-Ras4B1−166 6615


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Figure 9. Structural presentation of identified driver and anchor atoms for Ras. Hydrogen bonds between GppNHp/GDP and K-Ras4B are depicted by green dotted lines.

the catalytic domain. Backbone superimposition between the full-length K-Ras4B−GDP and the catalytic domain of H-Ras− GppNHp in complex with the Ras binding domain (RBD) of Raf (RafRBD) (PDB ID 4G0N)162 reveals that the HVR site partially overlaps the Ras effector binding region (Figure 8G).196 The different nucleotide-dependent orientation of the HVR with respect to the catalytic domain implies that interactions of the HVR with the catalytic domain are stronger for K-Ras4B−GDP than for K-Ras4B−GTP.197 Indeed, the measurement of binding affinity of a K-Ras4B HVR synthetic analog (Ac-KEKLNSKDGKKKKKKSKTK-NH2) with the KRas4B catalytic domain in the GDP- and GTP-bound states demonstrated that the interactions of the HVR with the KRas4B−GDP catalytic domain (Kd, 250.0 ± 33.4 nM) are much stronger by approximately 75-fold than those of the HVR with the K-Ras4B−GTP catalytic domain (Kd, 18.6 ± 0.9 μM).195 Previously, on the basis of NMR and isothermal titration calorimetry (ITC) experiments, Abraham et al.198 demonstrated that the HVR of K-Ras4B is the primary binding site for calmodulin (CaM), a ubiquitous Ca2+-binding protein, and that the affinity of K-Ras4B to CaM depends on the nucleotide binding to K-Ras4B. The studies showed little or no CaM binding to K-Ras4B in the GDP-bound form, suggesting that the HVR may be sequestered by the catalytic domain in KRas4B−GDP, preventing it from binding CaM. In contrast, in the GTP-γ-S-bound form, K-Ras4B is capable of interacting with CaM with micromolar affinity, pointing to an HVR disassociated state in K-Ras4B−GTP. These data suggest that in solution K-Ras4B−GDP is in its autoinhibited form via the interactions of the HVR with the catalytic domain, thereby locking K-Ras4B in an inactive state. Conversely, the exchange of GDP by GTP produces an active K-Ras4B−GTP featured by the release of HVR from the catalytic domain.197

and full-length K-Ras4B1−188 in the GDP- and GTP-γ-S-bound states.195 A comparison of the (1H, 15N) heteronuclear single quantum coherence (HSQC) spectra of GDP-bound KRas4B1−188 and K-Ras4B1−166 reveals that significant chemical shift perturbations (CSPs) are observed for a number of residues in the protein. The residues were mapped on the crystal structure of GDP-bound K-Ras4B catalytic domain and occur in β1 (L6), α1 (A18, I24), switch I (D33, T35-E37), β2 (Y40), L3 (G48, E49), β3 (L52), α2 (M72), β4 (L79), and α5 (A155, I163). Most of the residues that are perturbed significantly are in the switch I and effector binding regions, β2, and in the C-terminal helix α5. A comparison of the 1 H−15N HSQC spectra of GTP-γ-S-bound K-Ras4B1−188 and K-Ras4B1−166 reveals that significant differences of CSPs for backbone amides occur for residues in β1 (E3), α1 (A18), switch I (I36), β2 (V44), L3 (E49), β3 (T50, L52), β4 (F78, V81), and α3 (V103). On the basis of the NMR CSPs between the K-Ras4B1−188 and the K-Ras4B1−166 in its GDP-/GTP-γ-Sbound states, initial configurations of each full-length KRas4B−GTP/−GDP with a covalently connected HVR to His166 were generated through interactive molecular dynamics to relocate the HVR onto the catalytic domain. The conformation that has the lowest interaction energy between the HVR and the catalytic domain in the MD trajectory was selected as the optimal model for each full-length K-Ras4B− GDP/−GTP. In the starting point of the full-length K-Ras4B− GTP model, the multiple C-terminal lysine residues on the HVR interact with the negatively charged residues on the switch I region of the catalytic domain (Figure 8A,B). For example, residues K178, K180, K182, and K184 of the HVR form electrostatic interactions with residues E37, D38, D33, and E31 of the switch I region (Figure 8C). However, in the starting point of the full-length K-Ras4B−GDP model (Figure 8D,E), in addition to engagement of the switch I region, the HVR extends the electrostatic interactions with the switch II region of the catalytic domain. For example, residues K176, K178, K180, and K182 of the HVR form electrostatic interactions with residues E37 and D38 of the switch I and E62 of the switch II (Figure 8F). In the full-length K-Ras4B− GDP, the HVR runs between the L2 loop and the β2 strand of

3.4. Identified Driver and Anchor Atoms for Ras

Recently, we proposed a new concept of allosteric drug discovery. We suggested that allosteric drugs consist of anchor and driver atoms.81,199 The anchor atoms, by definition, occupy the allosteric site and are engaged in favorable interactions with the highly populated conformation in the inactive and active 6616


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structures with the exception of the N-Ras−GTP system, which was constructed using H-Ras−GTP as a template. A comprehensive comparison of the conformational dynamics of all three Ras isoforms showed that the significant differences in the dynamics of the isoforms were detected depending on the type of the nucleotide bound. For instance, in the 300 K simulations of GDP-bound states, the flexibility of the switch I region is in the order H-Ras > K-Ras4B > N-Ras, while the flexibility of the switch II region is in the order K-Ras4B > NRas > H-Ras. In contrast, in the 300 K simulations of GTPbound states, the flexibility of the switch I region is in the order K-Ras4B > H-Ras > N-Ras, while the flexibility of the switch II region is in the order K-Ras4B > N-Ras > H-Ras. Another interesting observation is the differential dynamics of the switch I region in the GDP- and GTP-bound states. The analysis showed that in both 300 and 360 K simulations of the H- and N-Ras, the switch I region is more dynamic in the GDP-bound state than in the GTP-bound state. This phenomenon has also been observed in previous MD simulations of H-Ras in the GTP-/GDP-bound states.207 In the 300 K simulations of KRas4B, the switch I region is more dynamic in the GDP-bound state than in the GTP-bound state, similar to H- and N-Ras. However, in the 360 K simulations, the switch I region of KRas4B is more dynamic in the GTP-bound state than in the GDP-bound state. The different conformational dynamics of Ras isoforms may relate to functional specificity.

states. These interactions between the anchor atoms of allosteric drugs and proteins do not undergo a change during the switch from the inactive to the active protein states and vice versa. In contrast, the driver atoms interact with proteins, and these interactions are profoundly different between the inactive and active states. Driver atoms can be further divided into attractive “pulling” or repulsive “pushing” atoms, depending on the specific interactions between the driver atoms and the corresponding residues/atoms in the allosteric sites. Guided by this concept, the formation of a stabilizing attractive interaction between driver atoms and proteins has the potential to pull the inactive conformation into the active conformation. Conversely, the formation of a repulsive interaction between driver atoms and proteins can push the active conformation into the inactive conformation.200 Ras proteins are activated by the exchange of GDP (Ras− GDP, inactive state) with GTP (Ras−GTP, active state). This exchange results in large conformational changes in the switch I and switch II regions of the Ras catalytic domains, resulting in Ras proteins in the active state that can bind to effector proteins to stimulate signaling pathways. The specific function of a protein rests on the extent to which a macromolecule populates its active conformation.201−203 Exploiting the anchor and driver identification protocol,81 it is applied to the catalytic domain of K-Ras4B in the GppNHp- (PDB ID 3GFT) and GDP-bound (PDB ID 4LPK) states. Two oxygen atoms from the γphosphate group of GppNHp are identified as driver atoms, which form hydrogen bonds with residue T35 from the switch I region and G60 from the switch II region (Figure 9). The two hydrogen bonds are absent in the GDP-bound structure. The pulling action from the two oxygen atoms of the γ-phosphate group of GppNHp may pull K-Ras4B from an inactive conformation to an active conformation and subsequently stabilize the Ras−GppNHp conformation. The anchor atoms are derived from the adenine, ribose, and α-/β-phosphate groups in both structures. These atoms interact with the inactive/active K-Ras4B via hydrogen bonds; these interactions do not change between the inactive and active states. For example, in both structures, the residues that interact with the anchor atoms include G13, G15, K16, S17, A18, V29, D30, D119, and A146.

5. CONFORMATIONAL PATH FROM THE ACTIVE RAS−GTP TO THE INACTIVE RAS−GDP On the basis of the crystal structures of GTP- and GDP-bound Ras, the switch I and switch II regions markedly change conformation in response to the release of γ-phosphate.58,208−211 The conformational changes in Ras that accompany the GTP hydrolysis are critical for offering insights into its function as a molecular switch in signaling pathways.156−160 5.1. Identifying the Conformational Path by Computational Methods

Nearly 2 decades ago, Ma and Karplus,216 for the first time, used the targeted molecular dynamics (TMD) method to investigate the conformational transition path from the active GTP-bound H-Ras to the inactive GDP-bound H-Ras. The wild-type GTP-bound H-Ras (PDB ID 5P21) and GDP-bound H-Ras (PDB ID 1Q21)150 were chosen as the two end-point structures, the former as the starting structure and the latter as the target structure. The GTP in the GTP-bound H-Ras was modified to GDP by removing the γ-phosphate to mimic the effect of the hydrolysis of the γ-phosphate. The TMD was subsequently performed in 200 ps by adding external forces to propel the conformational transition from the GTP-bound to the GDP-bound forms. In a similar vein, to explore the transition pathway from the GTP-bound to the GDP-bound states based on the same end-point structures, the nudged elastic band (NEB)217 method was carried out by inserting a string of replicas (or “images”) to form a discrete representation of a path from the start to the end point structures. Minimization of the entire system with the start and the end point structures fixed provides a minimum energy path. Both methods identified a similar conformational path. Comparing the conformational paths between the switch I and switch II regions during the transitions reveals that the transition of the switch I region is nearly completed before the

4. DIFFERENTIAL DYNAMICS OF THE CATALYTIC DOMAINS OF RAS ISOFORMS Despite the overall sequence and structural similarities shared by the catalytic domains of Ras isoforms, they may harbor distinct dynamics properties.204 On the basis of the crystal structure of H-Ras and two homology-built K-Ras4B and N-Ras with H-Ras as a template, classic MD simulations of the three Ras isoforms were performed in the nucleotide-free state.174 A comparative analysis of H-, N-, and K-Ras4B simulations showed that K-Ras4B is more dynamic than H- and N-Ras. Subsequently, using the crystal structures of K-Ras4B and HRas in the GTP-bound state, multicopy MD simulations were conducted by Grant et al.205 These revealed that K-Ras4B− GTP is intrinsically more flexible than H-Ras−GTP. This result is in good agreement with previous MD simulations of homology-built K-Ras4B and H-Ras in the nucleotide-free state.174 Recently, Kapoor and Travesset performed large-scale MD simulations of H-Ras, K-Ras4B, and N-Ras in both the GDP- (a total of 3.06 μs) and GTP-bound (2.4 μs) states.206 These simulations were deployed using the crystal structures of the three isoforms in complex with GTP and GDP as the initial 6617


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hydrolysis, the N-terminal switch II region moves away from the GDP and the C-terminal switch II region, namely, the helix α2 (residues 65−74), and subsequently undergoes a large rotation, coupled with an unwinding of a helical turn of Nterminal helix α2 (residues 65−69) (Figure 10). Significantly, during the transition of the switch II region from the GTP- to the GDP-bound state, transient hydrogen bonds between the carboxyl oxygen of E37 in the switch I region and the side chains of R68 and Y71 in the switch II region are observed (Figure 12B). The existence of these transient hydrogen bonds may be responsible for the stabilization of the intermediate where the helical turn at residues 65−69 unwinds. However, in both the starting GTP-bound (Figure 12A) and the ending GDP-bound (Figure 12C) states, no hydrogen-bonding networks are formed by all three residues. This result suggests that the conformation characterized by the formation of the transient hydrogen bonds among E37, R68, and Y71 may represent an intermediate during the GTP-to-GDP transition.

full transition in the switch II region has taken place (Figure 10), indicating that the transition of switch I region is more accessible than that of switch II region after the hydrolysis of γphosphate.

5.2. Capturing Intermediates in the Conformational Path by Crystallography

For wild-type Ras, it is difficult to determine the intermediates during the GTP-bound to the GDP-bound conformational transitions resulting from intrinsic GTP hydrolysis. However, it is possible to determine the unique conformational features of mutant Ras whose structure may resemble the intermediate in the conformational transition from Ras−GTP to Ras−GDP. 5.2.1. A59G H-Ras. The 1.70 Å high-resolution crystal structure of the A59G H-Ras mutant in complex with GppNHp was determined (PDB ID 1LF0).155 Backbone superimposition of the structure of H-RasA59G mutant onto that of wild-type HRas−GppNHp (PDB ID 5P21) shows that the switch I region incurs subtle conformational changes, while the switch II region undergoes extensive remodeling between the two structures (Figure 13A). This can be explained by A59, which resides at the beginning of the switch II region that connects the β3 strand and α2 helix and possesses high flexibility. Therefore, mutation of Ala59 to Gly would have a dramatic effect on the conformation of the switch II region. In particular, the helix α2 in the H-RasA59G mutant has undergone a rotation as compared with the wild-type H-Ras−GppNHp/GDP. Despite the relatively large conformational changes of the switch II region, the hydrogen bond between the G60 backbone NH group and the γ-phosphate of GppNHp is still maintained in the mutant (Figure 13B). Further superimposition of the crystal structure of wild-type H-Ras−GDP (PDB ID 4Q21) onto the two structures shows that the position of helix α2 in the H-RasA59G mutant is between its positions in the GppNHp- and GDPbound states of wild-type H-Ras (Figure 13A), which is indicative of an intermediate during the conformational transition from the wild-type Ras−GTP to wild-type Ras− GDP. To support this hypothesis, the intermediate captured by NEB simulations and the structure of H-RasA59G mutant were compared. Overall, for backbone atoms the RMSD between the two structures is 1.03 Å. Remarkably, the orientation of helix α2 in the two structures is extremely similar (Figure 13C). Furthermore, the relative positions of the three residues, E37, R68, and Y71, that form a transient hydrogen-bonding network observed in the intermediate from NEB simulations are also conserved in the two structures (Figure 13D). This similarity, together with the similarity of the position of helix α2, indicates that the structure of H-RasA59G−GppNHp mutant represents an intermediate during the conformational path from H-Ras−

Figure 10. Cartoon representation of the backbone superimposition of H-Ras structures derived from the beginning of the path (pink), the intermediate state (blue), and the ending of the path (cyan). Y32 in the switch I region and GDP are represented by stick models. The helical turn (residues 65−69) of N-terminal helix α2 in the beginning of the path is colored red.

In the switch I region, the largest conformational difference between the GTP- and GDP-bound states is in the orientation of the side chain of Y32.218 It is in the “up” conformation that points to the solvent in the GTP-bound state (Figure 11A). Conversely, in the GDP-bound state it moves to the interaction site where it resides in the “down” conformation and forms a hydrogen bond with the side chain of Y40 (Figure 11C). Inspection of the transition path of the switch I region shows an inward movement of the side chain of Y32 through a crevice between the switch I region and GDP, rather than an outward movement through space on the outside of the protein (Figure 11B). The motion of Y32 is coupled with the movement of four adjacent residues in the switch I region, E31, D33, E37, and D38 (Figure 11); these four negatively charged residues play a critical role in binding of RafRBD.162 In the starting GTPbound state, residues E31 and D33 of H-Ras form electrostatic interactions with residue K84 of RafRBD, and residues E37 and D38 of H-Ras form electrostatic interactions with residues R59, R67, and R89 of RafRBD (Figure 11D). In contrast, at the end of the GDP-bound state simulation, residues E31, D33, and E37 move away from the interaction site, resulting in the loss of electrostatic interactions. Simultaneously, the outward movement of D38 causes a steric conflict with the β-strand of RafRBD. Taken together, the conformational transition of the switch I region from the GTP- to the GDP-bound states renders GDP-bound H-Ras incapable of engaging with RafRBD, thereby turning off the signaling. Owing to the loss of the hydrogen bond between the G60 backbone NH group and the γ-phosphate after the GTP 6618


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Figure 11. Changes of the relative orientation of side chains of the E31, Y32, D33, E37, and D38 in the switch I region during the transition. GDP is depicted by sticks. (A) Beginning of the path in which the γ-phosphate of GTP is excluded. (B) Intermediate state. (C) Ending of the path in which the hydrogen bond between the side chains of Y32 and Y40 is depicted by a green dotted line. (D) Cartoon representation of the backbone superimposition of the crystal structure of H-Ras−GDP (PDB ID 4Q21) (cyan) onto that of H-Ras−GppNHp (pink) in complex with RafRBD (light blue) (PDB ID 4G0N). The two enlarged figures show the different interactions between the switch I residues and RafRBD in the GppNHp-/ GDP-bound H-Ras. In the starting GppNHp-bound state (pink), E31 and D33 form electrostatic interactions with residue K84 of RafRBD, and residues E37 and D38 form electrostatic interactions with residues R59, R67, and R89 of RafRBD. In the ending GDP-bound state (cyan), no electrostatic interactions are formed between residues E31, D33, and E37 of H-Ras and residues of RafRBD. Moreover, the steric conflict between D38 of H-Ras and the β-strand of RafRBD is observed.

Figure 12. Conformational changes of the switch II region (blue) during the transition. The changes of the relative orientation of side chains of the E37, R68, and Y71 are exhibited. GDP is depicted by stick models. (A) Beginning of the path in which no hydrogen bonds are formed among E37, R68, and Y71. (B) Intermediate state in which the transient hydrogen-bonding networks are formed among E37, R68, and Y71. The transient hydrogen bonds are depicted by green dotted lines. (C) Ending of the path in which there is only the formation of a hydrogen bond between E37 and R68.

the crystal structure of H-RasA59G−GppNHp, and wild-type HRas−GTP. The simulation of H-RasA59G−GDP was capable of sampling the GDP-bound conformation, suggesting a spontaneous conformational transition from Ras−GTP to Ras−GDP in the H-RasA59G mutant. To test this hypothesis, the crystal structure of H-RasA59G−GDP (PDB ID 1LF5)155 was superimposed on that of H-RasWT−GDP (PDB ID 4Q21). The comparison reveals that the two structures superpose well, with

GTP to H-Ras−GDP. Biochemical characterization of the HRasA59G mutant demonstrates that this mutant decreases the rate of intrinsic GTP hydrolysis by ∼10-fold and the rate of GppNHp dissociation by a 4-fold compared to the wild-type.155 To examine the influence of the A59G mutation on the conformational transition of H-Ras, Lukman et al.219 performed 20 ns unbiased MD simulations of H-RasA59G−GTP and HRasA59G−GDP, by removing the γ-phosphate of GppNHp from 6619


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5.2.2. Q61G H-Ras. Residue Q61 is located in the Nterminal switch II region and the 57DTAGQ61 motif plays an essential role in the cycling between the active GTP-bound and inactive GDP-bound forms of Ras.220 The 1.50 Å highresolution crystal structure of the Q61G H-Ras mutant in complex with GppNHp was determined (PDB ID 1ZW6).221 Backbone superimposition of the crystal structure of HRasQ61G−GppNHp onto that of H-RasWT−GppNHp shows that the switch I region in the Q61G mutant adopts a conformation similar to that observed in wild-type H-Ras, while the switch II region in the mutant undergoes a large conformational variation compared to the wild-type. This variation is ascribed to the enhanced flexibility of the switch II region induced by the mutation of Gln61 to Gly. Indeed, residues 61−64 of the N-terminal switch II region form an additional short 310 helix that extends helix α2 (residues 65− 75) by one turn in the H-RasQ61G−GppNHp mutant (Figure 15A). Further superimpositions of the crystal structure of H-

Figure 13. (A) Cartoon representation of the backbone superimposition of crystal structures of H-RasWT−GppNHp (PDB ID 5P21, pink), H-RasA59G−GppNHp (PDB ID 1LF0, orange), and H-RasWT− GDP (PDB ID 4Q21, cyan). (B) The formation of a hydrogen bond between the G60 backbone NH group and the γ-phosphate in the HRasA59G−GppNHp. (C) Backbone superimposition of the intermediate structure (blue) derived from MD simulations onto the crystal structure of H-RasA59G−GppNHp (orange). (D) The relative orientations of residues E37, R68, and Y71 between the intermediate structure (blue) and the crystal structure of H-RasA59G−GppNHp (orange).

a RMSD of 0.65 Å for the backbone atoms (Figure 14). Together, these results also indicate the existence of a lower energetic barrier between the GTP-bound and the GDP-bound states of the H-RasA59G mutant, which accelerates the conformational transition from Ras−GTP to Ras−GDP.

Figure 15. Cartoon representation of the backbone superimposition of crystal structures of H-RasWT−GppNHp (PDB ID 5P21, pink), HRasQ61G−GppNHp (PDB ID 1ZW6, blue), H-RasA59G−GppNHp (PDB ID 1LF0, orange), and H-RasWT−GDP (PDB ID 4Q21, cyan). The enlarged figure shows the conformations of the switch II region in these structures. (B) Conformational changes of residues E37, R68, and Y71 along the reaction path for the hydrolysis of GTP. Structures processed from the H-RasWT−GppNHp; to the intermediate 1, which is represented by the Q61G mutant (H-RasA59G−GppNHp); to the intermediate 2, which is represented by the A59G mutant (HRasQ61G−GppNHp); and finally to the H-RasWT−GDP. Hydrogen bonds are denoted by green dotted lines.

Figure 14. Cartoon representation of the backbone superimposition between the crystal structures of H-RasA59G−GDP (PDB ID 1LF5, yellow) and H-RasWT−GDP (PDB ID 4Q21, cyan). 6620


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Figure 16. (A) Cartoon representation of the backbone superimposition of crystal structures of inactive forms 1 (PDB ID 3KKN, blue) and 2 (PDB ID 3KKM, orange) of H-RasT35S−GppNHp onto that of active H-RasWT−GppNHp (PDB ID 5P21, pink). Residues Y35 and T/S35 and GppNHp are represented by stick models. (B) Both interactions of T35 and G60 with the γ-phosphate in the active H-RasWT−GppNHp. (C) The loss of both interactions of S35 and G60 with the γ-phosphate in the inactive form 1 of H-RasT35S−GppNHp. (D) Only G60 interacts with the γ-phosphate in the inactive form 2 of H-RasT35S−GppNHp. (E) Surface representation of the inactive form 1 of H-RasT35S−GppNHp. The positions of the two surface pockets found in the inactive form 1 of H-RasT35S−GppNHp are depicted by red open arrows. (F) Surface representation of the active state of H-RasWT−GppNHp. (G) Surface representation of the inactive form 2 of H-RasT35S−GppNHp. (H) The inactive form 3 of K-Ras4BWT−GTP derived from MD simulations, which is characterized by only the interaction of T35 with the γ-phosphate. (I) Surface representation of the inactive form 3 of K-Ras4BWT−GTP. Hydrogen bonds are depicted by green dotted lines.

RasQ61G−GppNHp onto those of H-RasWT−GppNHp (the beginning of the path), H-RasA59G−GppNHp (intermediate), and H-RasWT−GDP (the end of the path) show that helix α2 in the H-RasQ61G−GppNHp mutant is at its position between the GppNHp- and GDP-bound states of H-RasWT. More precisely, positionwise, its structure is between those of H-RasWT− GppNHp and H-RasA59G−GppNHp (Figure 15A). This suggests that the structure of H-RasQ61G−GppNHp represents another transient intermediate of H-Ras on the reaction path following GTP hydrolysis. TMD simulations were subsequently carried out on the structure of H-RasQ61G−GppNHp to explore the conformational path from the GTP-bound to the GDPbound states. The simulations revealed that the transient hydrogen-bonding network involving E37, R68, and Y71 is characteristic of the intermediate observed in the H-RasQ61G mutant.155,221 Further TMD simulations were conducted using the structures of H-Ras WT −GppNHp and H-RasA59G − GppNHp as the starting and target structures, respectively. Analysis of the TMD-trajectory showed that the features of HRasQ61G−GppNHp were observed in the TMD simulation snapshots. These results imply that the structure of H-RasQ61G−

GppNHp represents an intermediate after the GTP hydrolysis, but preceding the structures of H-RasA59G−GppNHp and HRasWT−GDP. As a result, two intermediates, H-RasA59G− GppNHp and H-RasQ61G−GppNHp, were identified on the conformational path from Ras−GTP to Ras−GDP (Figure 15B).

6. CONFORMATIONAL ENSEMBLE OF RAS−GTP IN SOLUTION 6.1. Ras−GTP Exists in Active and Inactive States

Previous 31P NMR spectroscopic studies of wild-type H-Ras catalytic domain in complex with GppNHp and Mg2+ entailing comparisons of chemical shift changes for the resonance of the nucleotide phosphorus atoms of the α-, β-, and γ-phosphates showed that H-RasWT−GppNHp exists in two distinct conformational states, inactive state 1 and active state 2.222,223 The equilibrium constant K between the two states of wild-type H-Ras is close to 1 and the interconversion between the two states occurs on a millisecond time scale. Mutational experiments further indicated that mutations in the switch II region (E62H or S65P) have no significant effects on the equilibrium, 6621


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Figure 17. Different states of GTP-bound Ras in solution described by the coupling interactions between γ-phosphate and the switch I and switch II regions involving the side chain oxygen atom of T35 (colored red in the switch I) and the backbone nitrogen atom of G60 (colored blue in the switch II). The P-loop, switch I, and switch II regions are colored lime, pink, and blue, respectively. The active state of Ras is characterized by the stabilization of the switch I and switch II by the GTP through interaction of T35 and G60 with the γ-phosphate. Surface representation of the active state of H-Ras (PDB ID 3K8Y) shows that the three functional P-loop, switch I, and switch II regions form the closed conformation of the GTP binding site. The inactive substate 1 of Ras is characterized by the disassociation of both T35 and G60 from the γ-phosphate. Surface representation of the inactive substate 1 of H-Ras (PDB ID 3KKN) shows the wide open conformation of the GTP binding site. The inactive substate 2 of Ras is characterized by only the loss of interaction of T35 with the γ-phosphate. Surface representation of the inactive substate 2 of H-Ras (PDB ID 3KKM) shows the semiopen conformation of the GTP binding site. The inactive substate 3 of Ras is only identified from MD simulations, which is characterized by only the loss of interaction of G60 with the γ-phosphate. The surface representation of the inactive substate 3 of K-Ras4B shows the semiopen conformation of the GTP binding site.

the interaction site where it is in the “down” conformation in the inactive form 1 of H-RasT35S−GppNHp. Further comparative analysis of the interactions of residue T35 in the switch I region and residue G60 in the switch II region with the γphosphate indicates that the active state is characterized by the stabilization of the switch I and switch II through interaction of T35 and G60 with the γ-phosphate (Figure 16B), with the inactive form 1 described by disassociation of both T35 and G60 from the γ-phosphate (Figure 16C) and the inactive form 2 featured by only the loss of interaction of T35 with the γphosphate (Figure 16D). Due to the extensive remodeling of the switch I region, two surface pockets occur in the inactive form 1. One pocket, designated as pocket 1, is located between the two switch regions, and the other pocket, designated as pocket 2, is located between the switch I flanking residues 28− 31 and GppNHp (Figure 16E). The two pockets, however, do not exist in the active state of H-RasWT−GppNHp (Figure 16F). Similarly, pocket 2 observed in the inactive form 1 does not occur in the inactive form 2 (Figure 16G). However, the existence of an equivalent pocket 1 of the inactive form 1 in the inactive form 2 remains unclear owing to the completely invisible electron density of the switch II residues 61−71 in the

while mutations in the P-loop (G12D) or the switch I region (Y32R or T35A) have dramatic effects on the equilibrium, which shift the population to the inactive state 1.223 Through crystallization of T35S H-Ras in complex with GppNHp, Shima et al.153 determined the first 3D structure of the inactive state 1 of H-Ras−GppNHp. Two distinct structures (inactive forms 1 and 2) corresponding to the inactive state 1 were obtained. The electron density of the switch I and switch II regions is completely visible in the inactive form 1, whereas that of switch II residues 61−71 is invisible in the inactive form 2. Backbone superimposition of the crystal structures of the inactive forms 1 (PDB ID 3KKN)153 and 2 (PDB ID 3KKM)153 onto that of GppNHp-bound H-Ras (PDB ID 5P21) shows that the switch I and switch II regions of the two inactive forms of H-Ras T35S −GppNHp display marked deviations from the active state of H-RasWT−GppNHp (Figure 16A). Inspection of the two switch regions exhibits that the switch I region in the inactive form 1 undergoes a larger reorganization than in the inactive form 2. For example, Y32 is in the “up” conformation in H-RasWT−GppNHp; it shifts to the crevice between the switch I region and GppNHp in the inactive form 2 of H-RasT35S−GppNHp, and finally it moves 6622


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Figure 18. (A) Cartoon representation of the backbone superimposition of inactive state 1 crystal structures of H-RasWT−GppNHp (PDB ID 4EFL, yellow), H-RasG12V−GppNHp (PDB ID 4EFM, orange), and H-RasQ61L−GppNHp (PDB ID 4EFN, green) onto the crystal structure of inactive form 1 of H-RasT35S−GppNHp (PDB ID 3KKN, blue). Residues G60 and T/S35 and GppNHp are represented by stick models. Surface representation of the inactive state 1 of H-RasWT−GppNHp (B), H-RasG12V−GppNHp (C), and H-RasQ61L−GppNHp (D). The positions of the two surface pockets are depicted by red open arrows.

substates in solution and the energy for the different states of Ras−GTP follows the order active state > inactive form 3 > inactive form 2 > inactive form 1 (Figure 17). Recently, Muraoka et al.224 solved the inactive state 1 crystal structures of H-RasWT−GppNHp (PDB ID 4EFL) together with its oncogenic G12V (PDB ID 4EFM) and Q61L (PDB ID 4EFN) mutants, which, for the first time, unequivocally ascertain the existence of inactive and active states in the WT and oncogenic mutants of Ras. Detailed inspection of interactions of T35 and G60 with the γ-phosphate shows that both the T35−γ-phosphate and G60−γ-phosphate interactions are lost in all three structures (Figure 18A), which resemble the inactive form 1 of H-RasT35S−GppNHp.153 The backbone superimposition of WT, G12V, and Q61L structures onto the inactive form 1 of H-RasT35S−GppNHp points to subtle conformational changes between each structure (Figure 18A). Interestingly, the two surface pockets, pockets 1 and 2 observed in the inactive form 1 of H-RasT35S−GppNHp, are also found in the inactive state 1 of the WT (Figure 18B), G12V (Figure 18C), and Q61L (Figure 18D) structures. However, the shapes of the two surface pockets in the G12V and Q61L mutants are slightly different from those in the WT, resulting from local conformational changes triggered by mutations, which could be harnessed in structure-based drug design that specifically targets the activated oncogenic mutants.

inactive form 2, which consist of a main part of the pocket 1 in the inactive form 1. MD simulations of K-Ras4BWT−GTP also showed the existence of both active and inactive states, which argues that the conformational equilibrium between the two states may be common across members of the small GTPase family in their GTP-bound forms. The active state of K-Ras4B from the simulation resembles the active state of H-Ras characterized by both interactions of T35 and G60 with the γ-phosphate. However, only the lack of the interaction of G60 with the γphosphate, referred to as inactive form 3 (Figure 16H), was observed in the inactive state of K-Ras4B, which is distinct from the two inactive forms of H-Ras described by both the uncoupling of T35 and G60 from the γ-phosphate (Figure 16C) or only the uncoupling of T35 from the γ-phosphate (Figure 16D). Analysis of surface pockets in the inactive form 3 of K-RasWT−GTP showed that an equivalent pocket 2 of inactive form 1 does not occur in the inactive form 3, whereas an equivalent pocket 1 of inactive form 1 can be observed in the inactive form 3 (Figure 16I), although the volume of this pocket in the inactive form 3 is smaller than that in the inactive form 1. However, the conformation of K-Ras4BWT−GTP does not visit the two higher energy inactive forms, 1 and 2, in the short MD time scales. The difference between the structures of the inactive form 3 of K-Ras4BWT−GTP and the two inactive forms 1 and 2 of H-RasT35S−GppNHp is attributed to the observation that in H-Ras the T35 that is coordinated to Mg2+ was mutated to S35, which effectively captures large conformational changes in the switch I region. Collectively, MD simulations, coupled with the crystallographic data, suggest that the inactive state of Ras−GTP may exist in at least three

6.2. Oncogenic Mutations Shift the Ras−GTP Ensemble toward the Active State

MD simulations of K-Ras4BWT−GTP revealed that the conformational ensemble of K-Ras4BWT−GTP contains two major active and inactive conformers. The two states are described by probability distributions for two atom-pair 6623


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Figure 19. Probability distributions for two atom-pair distances, d1 (defined by the distance from the G60 Cα atom to the GTP Pβ atom) and d2 (defined by the distance from the T35 Cα atom to the GTP Pβ atom), were calculated on the MD snapshots of K-Ras−GTP and converted into the surface plot representing the potential of mean force (PMF). (A) wild-type, (B) G12C, (C) G12D, (D) G12V, (E) G13D, and (F) Q61H mutants. The inset figure in the panel B illustrates the definition of d1 and d2 in K-Ras4B−GTP.

inhibitors (FTIs) have been reported to inhibit membrane localization of Ras proteins by blocking downstream Ras signaling.225 However, the antitumor activity of FTIs failed due an alternative PTM of Ras, including prenylation and geranylgeranylation, escaping the FTIs’ inhibition. An antibody can specifically inhibit oncogenic Ras through interaction of single variable region (V) domains (iDabs) with activated Ras−GTP mutants.226 Tanaka et al.227 showed that a single domain antibody fragment, iDab#6, is capable of binding oncogenic H-Ras−GTP with mutations at either G12 or Q61, and the binding affinity of iDab#6 to the oncogenic mutant Ras is 2 orders of magnitude stronger than to H-Ras−GTPWT. IDab#6 recognizes a conformational epitope on H-Ras−GTP. Mutations at the switch I and switch II regions, including D33E, P34L, T35S, D38E, Y40C, and Y64G, markedly reduced the association with iDab#6. These data indicate that iDab#6 interacts with activated H-Ras−GTP via the switch I and switch II regions. Further comparison of the binding affinity of iDab#6 with both H-Ras−GTPWT and H-Ras−GTPG12V in their GTPand GDP-bound states revealed that the intrabody, iDab#6, interacts specifically with the active state of H-Ras−GTP, rather than the inactive state of H-Ras−GDP. Further expressing the intrabody in cells harboring oncogenic mutant H-RasG12V, the results showed that the anti-Ras intrabody, iDab#6, is able to inhibit tumorigenesis and metastasis in a mouse model.227 The 2.0 Å resolution X-ray crystal structure of H-RasG12V− GTP in complex with the anti-Ras intrabody in an Fv format was solved (PDB ID 2UZI).227 The anti-Ras Fv consisted of the VH#6 plus an anti-Ras VL isolated from a synthetic VL intrabody library. The 3D structure of the H-RasG12V−GTP− anti-Ras Fv complex reveals that the VH and VL domains interact mainly with the switch I and switch II regions of H-

distancesone is defined by the distance from the Cα atom of switch II residue G60 to the Pβ atom of GTP (d1) and the other from the Cα atom of switch I residue T35 to the Pβ atom of GTP (d2)and converted into a surface plot representing the potential of mean force (PMF). For K-Ras4BWT−GTP, two energy-minima basins are observed (Figure 19A); one is the active state and the other the inactive state. To explore the effects of oncogenic mutations on the conformational ensemble of K-Ras4B−GTP, the probability distributions representing the PMF for each oncogenic mutant were calculated for the distances of the same atom-pairs. Compared to the surface plot of K-Ras4BWT−GTP (Figure 19A), the conformers of oncogenic mutants predominantly exist in an active state, especially the G12D (Figure 19C), G12V (Figure 19D), G13D (Figure 19E), and Q61H (Figure 19F) mutants. However, the conformational ensemble of the G12C mutant is similar to that of the wild-type one, suggesting that the G12C mutant still exists in two, active and inactive, states (Figure 19B). This observation suggests that the oncogenic mutations, except the G12C mutation, cause an inactive-to-active conformational transition in the mutant K-Ras4B−GTP, which results in a higher population of active K-Ras4B−GTP in the oncogenic mutants than in the wild-type. 6.3. Anti-Ras Intrabody Inhibits the Active State of Ras−GTP

The active state of Ras−GTP or mutation-activated Ras−GTP can bind its downstream effectors, such as Raf, PI3K, and RalGDS, to initiate downstream signaling cascades, which contribute to the development of cancer. Thus, directly inhibiting activated Ras−GTP mutants is a promising approach in human cancer therapy. A number of farnesyltransferase 6624


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Figure 20. (A) Cartoon representation of the crystal structure of the H-RasG12V−GTP−anti-Ras single domain complex (PDB ID 2UZI). HRasG12V−GTP is shown in gray, the switch I region in pink, and the switch II region in blue. The Fv proteins VH and VL are shown in orange and green, respectively. GTP is depicted by stick models. Stereo representation of the backbone superimposition of crystal structures of H-RasWT− GppNHp−RafRBD (PDB ID 4G0N, RafRBD in cyan) (B), H-RasE31K−GppNHp−RalGDS-RBD (PDB ID 1LFD, RalGDS-RBD in magenta) (C), and H-RasG12V−GppNHp−PI3Kγ-RBD (PDB ID 1HE8, PI3Kγ-RBD in red) (D) onto that of H-RasG12V−GTP−anti-Ras single domain complex. H-Ras protein in the Ras−effector complexes is hidden for clarity.

Figure 21. Chemical structures of Kobe0065 (A), Kobe2602 (B), and Kobe2601 (C). Surface (D) and cartoon (E) representations of the lowest energy NMR structure of H-RasT35S−GppNHp−Kobe2601 complex (PDB ID 2LWI). Kobe2061 and GppNHp are shown by stick models. Residues S35 and G60 form no interactions with the γ-phosphate.

RasG12V−GTP, respectively (Figure 20A). Backbone superimposition of the crystal structure of H-RasG12V−GTP−antiRas Fv onto those of H-RasWT−GppNHp−RafRBD (PDB ID 4G0N) (Figure 20B), H-RasE31K−GppNHp−RalGDS-RBD (PDB ID 1LFD) (Figure 20C), and H-RasG12V−GppNHp− PI3Kγ-RBD (PDB ID 1HE8) (Figure 20D) reveals that the intrabody significantly overlaps the Ras effector binding region, suggesting that the anti-Ras intrabody competes with the Ras effectors and inhibits the Ras−effector interactions, thereby interfering with Ras-associated signal transduction pathways. In support of this notion, the binding affinity of the anti-Ras intrabody was measured using purified protein by surface resonance plasma. The Kd of the Ras−intrabody complexes is ∼0.4 nM for scFV#6 (including both VH#6 and VL#6) to HRasG12V−GTP-γ-S and 6 nM for VH#6 to H-RasG12V−GTP-γS,227 which are lower than the 18 nM Kd of H-Ras−RafRBD,194 1 μM Kd for H-Ras−RalGDS-RBD,228 and 3.2 μM Kd for HRas−R PI3Kγ complexes.163 This mode of intrabody action is a novel advance to directly interfere with oncogenic Ras function in human cancer.

6.4. Compounds Stabilize the Inactive State of Ras−GTP

Given that the conformational ensemble of Ras−GTP contains two major active and inactive conformers and the inactive form 1 renders two surface pockets that are not seen in the active state, it offers promising opportunities for structure-based drug design with small-molecule compounds that directly target the two pockets of the inactive state and subsequently shift the population to the inactive state, which is predicted to inhibit Ras function. 6.4.1. Kobe0065 Family Compounds. On the basis of the unique pocket 1 observed in the inactive form 1 of H-Ras− GppNHp, Shima et al.70 performed a structure-based virtual screening of a library consisting of 40 882 compounds. The molecular mechanics Poisson−Boltzmann surface area (MMPBSA) binding free energy calculations were carried out to evaluate potential binding between the candidates and the protein. Ninety-seven candidates were chosen and their abilities to inhibit the H-Ras−GTP−RafRBD complex were examined. Only one compound, Kobe0065 (Figure 21A), was found to competitively inhibit the binding of H-Ras−GTP to RafRBD, with a Ki value of 46 ± 13 μM. A similarity search of Kobe0065 6625


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was performed on three libraries containing ∼160 000 compounds, and 273 compounds were selected on the basis of Tanimoto coefficients of ≥0.7 between Kobe0065 and each of these. One compound, Kobe2602 (Figure 21B), was identified with a Ki value of 149 ± 55 μM. Owing to the low water solubility of Kobe0065 and Kobe2602, another watersoluble compound identified from the similarity search, Kobe2601 (Figure 21C), was tested with respect to its ability to recognize the protein−ligand binding site using NMR spectroscopy. Only a relatively weak inhibitory activity of Kobe2601 against H-Ras−RafRBD complex was measured, with a Ki value of 779 ± 49 μM. The solution NMR structures of H-Ras T35S −GppNHp−Kobe2601 complex (PDB ID 2LWI)70 showed that Kobe2601 is located between the two switch regions (Figure 21D), which is similar to the position of the pocket 1 observed in the crystal structure of H-RasT35S− GppNHp.153 Further analysis of the interactions of S35 and G60 with the γ-phosphate observed disengagement of both S35 and G60 from the γ-phosphate (Figure 21E), supporting the notion that Kobe2601 binds to the inactive state of H-RasT35S− GppNHp and subsequently shifts the ensemble to this state. Functional characterization of the Kobe0065 family of compounds showed that they display antiproliferative activity on a xenograft of human colon carcinoma SW480 cells carrying the K-RasG12V oncogene. This study suggests the potential to exploit the surface pockets in the unique conformation of Ras as new target sites for Ras inhibitors and provides an opportunity to design Ras inhibitors with higher potency and specificity exploiting the Kobe0065 family of compounds as a scaffold. 6.4.2. Zn2+/Cu2+−Cyclen Complexes. Ras−GTP exists in two distinct conformational states, inactive state 1 and active state 2, which are in dynamic equilibrium.229−232 The binding affinities of the two states to their effectors, however, are remarkably different. The affinity of state 2 for effectors is 2 orders of magnitude larger than that of state 1.99,102 Thus, state 2 represents a strong binding state for effectors, while state 1 represents a weak binding state for effectors. In this regard, selective stabilization of the weak effector-binding state 1 by small-molecule compounds is gaining increasing recognition as a promising novel strategy for the interruption of oncogenic Ras signaling. 1,4,7,10-Tetraazacyclodecane, cyclen, has been well-established as a macrocyclic ligand for complexation with various transition-metal cations, such as Zn2+ and Cu2+, providing useful platforms for recognizing proteins, receptors, and supramolecules.234 31P NMR spectroscopy showed that after addition of Zn2+−cyclen (Figure 22A) or Cu2+−cyclen (Figure 22B) to H-RasWT−GppNHp, Zn2+/Cu2+−cyclen selectively bind to state 1 of H-Ras−GppNHp and subsequently shift the conformational ensemble of H-Ras−GppNHp toward the weak binding state 1.235,236 Comparison of the CSPs from 1H−15N HSQC NMR spectra reveals two distinct sites for Cu2+/Zn2+− cyclen on the surface of H-RasWT. The first binding site is proximal to the γ-phosphate, as observed by the significantly perturbed residues from G13, Y32, and A59−Q61. The second binding site is predicted to be near the L7 loop containing residues D105−V109 and M111 and the C-terminal helix α5 containing residues E162, Q165, and H166. X-ray crystallography was then performed to confirm the two binding sites of Zn2+/Cu2+−cyclen. The 2.1 Å crystal structure of H-RasWT− GppNHp in complex with Zn2+−cyclen was obtained (PDB ID 3L8Y).235 Unfortunately, binding site 1 is not captured in the

Figure 22. Chemical structures of Zn2+−cyclen (A) and Cu2+−cyclen (B). (C) Cartoon representation of the crystal structure of H-RasWT− GppNHp−Zn2+−cyclen complex (PDB ID 3L8Y). The binding site 2 of Zn2+−cyclen is located close to loop L7 and the C-terminal helix α5.

crystal structure. Only binding site 2, which is located close to the L7 loop and the C-terminus, is observed (Figure 22C), which is in good agreement with the NMR data. ITC was conducted to evaluate the H-Ras/RafRBD binding affinity in the presence of Zn2+/Cu2+−cyclen complexes. As expected, the results showed that the binding affinity of RafRBD to HRasWT−GppNHp decreases markedly in response to either Zn2+−cyclen or Cu2+−cyclen, supporting the notion that Zn2+− cyclen or Cu2+−cyclen binds to the weak binding state 1 of HRas WT−GppNHp. Similar to H-Ras, recent studies by Rosnizeck et al.237 have also revealed that Zn2+−cyclen interacts selectively with the weak binding state 1 of KRas4B. Taken together, these data suggest that Zn2+−cyclen or Cu2+−cyclen can function as a lead compound for interrupting the Ras−effector interactions, thereby turning off Ras signaling. 6.4.3. Zn2+−BPA Complexes. Zn2+−bis(2-picolyl)amine (Zn2+−BPA) (Figure 23A) is an alternative class of state 1 stabilizers of Ras−GTP.238 By analogy with the observations with Zn2+/Cu2+−cyclen complexes, 31P NMR spectroscopy also unequivocally ascertained that Zn2+−BPA selectively interacts with state 1 of H-RasWT−GppNHp and subsequently shifts the conformational ensemble of H-Ras−GppNHp toward the weak binding state 1.238 1H−15N HSQC NMR spectra, coupled with the 31P NMR data, indicated the existence of two distinct binding sites of Zn2+−BPA on the surface of H-RasWT− GppNHp. One binding site of Zn2+−BPA is located in the vicinity of residues D38−Y40, which is outside the nucleotidebinding pocket. This binding site of Zn2+−BPA is different from the binding site 1 of Zn2+−cyclen; the latter is located proximal to the active center (Figure 23B). The second binding site of Zn2+−BPA is located at loop L7 and the C-terminal helix α5, which is similar to the binding site 2 of Zn2+−cyclen observed by X-ray crystallography.235 However, the crystal structure of H-RasWT−GppNHp−Zn2+−BPA is still unresolved. As a result, whether Zn2+−BPA binds to the two mentioned binding sites predicted by 35P NMR and 1H−15N HSQC NMR spectroscopy remains unclear. Although both Zn2+−BPA and 6626


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7. STABILIZATION OF RAS−GDP BY S-IIP BINDING COMPOUNDS Ras functions as a molecular switch cycling between inactive GDP-bound and active GTP-bound states. The conversion of inactive Ras−GDP to active Ras−GTP consists of two steps, GDP dissocation and incorporation of a new molecule of GTP. GDP dissocation from the Ras active site is the rate-limiting step in Ras activation. In this regard, designing compounds that disrupt this conversion and subsequently stabilize Ras in its inactive GDP-bound state affords an attractive therapeutic strategy aimed at inhibiting Ras activity for the treatment of cancer. Oncogenic K-RasG12C mutant harbors an introduced cysteine residue in the P-loop. The mutant cysteine residue makes it possible to design covalent inhibitors that are formed through a disulfide bond between SH-containing compounds and the cysteine. This site-directed technology, known as disulfide trapping (or tethering),239 provides a straightforward strategy to detect and characterize undiscovered binding sites in proteins of interest.240 On the basis of K-Ras4BG12C, Ostrem et al.73 recently took advantage of a disulfide-fragment-based screening approach to screen a library of 480 disulfide-containing small-molecule compounds. They found that two fragments, 6H05 (Figure 24) and 2E07 (Figure 24), led to significant modification of KRas4BG12C without affecting wild-type K-Ras4B. Further modification of 6H05 identified compound 6 (Figure 24), which displayed the greatest potency in inhibiting K-Ras4BG12C enzymatic activity. The determination of the X-ray cocrystal structure of K-Ras4BG12C in complex with compound 6 (PDB ID 4LUC) revealed that, as expected, compound 6 forms a disulfide bond with C12 (Figure 25A). The binding site of compound 6 is located between the central sheet β1, the switch II region, and helix α3, which does not overlap with the nucleotide-binding site (Figure 25B). Backbone superimposition of K-Ras4BG12C−GDP−compound 6 structure onto KRas4BG12C−GDP unbound structure (PDB ID 4L9S) and KRasWT−GDP structure (PDB ID 4LPK) shows that the binding site of compound 6 beneath the effector binding switch II region, termed S-IIP, is not observed in the latter two crystal

Figure 23. (A) Chemical structure of Zn2+−BPA. (B) The locations of the binding site 1 of Zn2+−BPA (red) and Zn2+−cyclen (magenta) on the surface of the inactive state 1 crystal structure of H-RasWT− GppNHp (PDB ID 4EFL).

Zn2+−cyclen can bind to the weak binding state 1 of H-RasWT− GppNHp, their binding abilities to the oncogenic Ras mutants are significantly different. As in the case of H-RasG12V oncogenic mutant, Zn2+−BPA, at a concentration of 5 mM, is able to completely shift the conformational ensemble of H-RasG12V− GppNHp to state 1, while this effect cannot be completely accomplished by Zn2+−cyclen, even at a 25 mM higher concentration. Thus, the association of Zn2+−BPA with HRasG12V−GppNHp is much stronger than that of Zn2+−cyclen. This indicates that Zn2+−BPA represents a better lead compound than Zn2+−cyclen for inhibiting the oncogenic Ras−effector interactions.

Figure 24. Chemical structures of disulfide-based compounds, 6H05, 2E07, and compound 6, as well as carbon-based compounds, vinyl sulphonamides (compound 8) and acrylamides (compounds 11 and 12). 6627


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Figure 25. Cartoon (A) and surface (B) representations of the crystal structure of K-Ras4BG12C−GDP−compound 6 complex (PDB ID 4LUC). Compound 6 forms a disulfide bond with C12. The binding site of compound 6 is lined by the central sheet β1, switch II region, and helix α3. (C) Backbone superimposition of the crystal structure of K-Ras4BG12C−GDP−compound 6 (pink) onto those of K-Ras4BG12C−GDP (PDB ID 4L9S, orange) and K-Ras4BWT−GDP (PDB ID 4LPK, cyan). (D) Surface representation of the crystal structure of K-Ras4BG12C−GDP−compound 8 complex (PDB ID 4LYF). (E) Surface representation of the structure of K-Ras4BG12C−GDP−compound 11 complex (PDB ID 4M21). (F) Backbone superimposition of the crystal structure of K-Ras4BG12C−GDP−compound 6 (pink) onto that of K-Ras4BG12C−GDP−compound 8 (PDB ID 4L9S, cyan).

Figure 26. (A) Cartoon representation of the backbone superimposition of the crystal structure of H-RasWT−GppNHp−Ca(OAc)2 (PDB ID 3K8Y, pink) onto that of H-RasWT−GppNHp−CaCl2 (PDB ID 2RGE, lime). (B) The detailed interactions between Ca(OAc)2 and H-Ras. The coordination interactions and hydrogen-bonding interactions are depicted by blue and green dotted lines, respectively. (C) The active site of HRasWT−GppNHp−Ca(OAc)2 showing Y32, Q61, and the catalytic (W175) and bridging (W189) water molecules near the nucleotide. (D) Hydrogen-bonding network 1 centered on R97. (E) Hydrogen-bonding network 2 centered on R68. (F) Proposed mechanism of intrinsic GTP hydrolysis in H-Ras. 6628


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acetate, three oxygen atoms from water molecules (W82, W392, W366), and two backbone carbonyl oxygen atoms of D107 on loop L7 and Y137 on helix α4. In addition, the two oxygen atoms of acetate form hydrogen bonds with the side chain of R97 on helix α3 and the backbone NH group of V109 on loop L7, respectively. These observations suggest that the interactions of Ca(OAc)2 with residues in helix α3, loop L7, and helix α4 trigger conformational changes in the allosteric lobe of H-Ras that can transmit to the effector lobe to order the switch II region.241 Under these circumstances, the catalytic residue Q61 in the ordered switch II region can interact with the bridging water molecule (W189), which in turn interacts with the side chain of Y32 and γ-phosphate (Figure 26C), rendering the H-Ras−GTP in its precatalytic conformation at the beginning of the hydrolysis reaction. In this context, the HRasWT−GppNHp−Ca(OAc)2 structure represents a catalytically competent state (“on” conformation), while the HRasWT−GppNHp−CaCl2 structure represents a catalytically incompetent state (“off” conformation). A comparative analysis between the structures of H-RasWT− GppNHp−Ca(OAc)2 and H-RasWT−GppNHp−CaCl2 identifies the existence of two hydrogen-bonding networks in the HRasWT−GppNHp−Ca(OAc)2 structure that connect the allosteric site to residue Q61. Network 1 contains the Ca(OAc)2, which is centered on R97. This network is formed by residues from helix α3 (H94, R97, E98, and K101), loop L7 (D107), and helix α4 (Y137) (Figure 26D). Network 2 is centered on R68 and includes mainly the water-mediated hydrogen-bonding interactions that propagate from Y96 (W367) and Q99 (W384) on the helix α3 to A59 (W373) and Q61 (W372) on the switch II region (Figure 26E). The two networks contribute to order the switch II region with the alignment of the catalytic residue Q61 adjacent to the bridging water molecule W189. In the active site, the catalytic water molecule W175 associates with the γ-phosphate and the bridging water molecule W189 interacts with Y32, Q61, and the γ-phosphate (Figure 26C). On the basis of these observations, the mechanism of intrinsic GTP hydrolysis has been proposed (Figure 26F). A proton from the catalytic water molecule W175 is shuttled to the bridging water molecule W189 through the O1G atom of the γ-phosphate of GTP. The bridging water molecule W189 donates two hydrogen bonds via the side chain hydroxyl group of Y32 and the side chain carbonyl group of Q61. The developing negative hydroxide ion can attack the positively charged γ-phosphate phosphorus atom of GTP to produce GDP and inorganic phosphate.

structures (Figure 25C). This observation suggests that compound 6 binds to a new allosteric pocket of KRas4BG12C−GDP. Examination of the superimposed structures reveals that compared with the structures of K-Ras4BG12C− GDP and K-Ras4BWT−GDP, the switch II region in the KRas4BG12C−GDP−compound 6 structure undergoes extensive restructuring, and the conformational change of the switch I region is not obvious. To improve on the disulfide-based compounds, the authors used carbon-based electrophiles, vinyl sulphonamides (compound 8, Figure 24) and acrylamides (compounds 11 and 12, Figure 24), to modify residue C12 of K-Ras4B. The determination of X-ray cocrystal structures of KRas4BG12C in complex with the corresponding compounds 8 (PDB ID 4LYF) (Figure 25D) and 11 (PDB ID 4M21) (Figure 25E) apparently revealed that both compounds, located in SIIP, are involved in a disulfide bond with C12. Backbone superimposition between the K-Ras4BG12C−GDP−compound 6 structure and the K-Ras4BG12C−GDP−compound 8 structure shows that compared with compound 6, compound 8 induces more marked conformational changes of the switch II region, which in turn led to disordering of the switch I region (Figure 25F). A plate-based assay of binding affinities of GTP and GDP to K-Ras4BG12C in the presence or absence of S-IIP binding compounds was performed to explore the impact of S-IIP binding compounds on the nucleotide affinities of K-Ras4B. The results revealed that in the absence of S-IIP binding compounds, the binding affinity of GTP to K-Ras4BG12C is slightly stronger than that of GDP to K-Ras4BG12C; in striking contrast, the binding affinity of GTP to K-Ras4BG12C is significantly decreased compared to that of GDP to KRas4BG12C in response to binding of S-IIP binding compound 8 or 12. Furthermore, compounds 8 and 12 disrupt the exchange factor SOS catalyzed nucleotide exchange. These results suggest that binding of S-IIP binding compounds shifts the conformational ensemble of K-Ras4B to the inactive GDPbound state and subsequently stabilizes this state. Together, this study uncovers a new allosteric regulatory site on K-Ras4B that can be targeted in a mutant-specific manner and can serve as a starting point in the design of allosteric modulators.

8. ALLOSTERIC SWITCH OF RAS−GTP TO THE ON STATE Previously, Buhrman et al.158 solved the crystal structure of HRasWT−GppNHp that was grown in 200 nM calcium chloride (CaCl2) and found that the switch II region is disordered with no electron density for the catalytic residue Q61 (PDB ID 2RGE). Replacing CaCl2 by calcium acetate [Ca(OAc)2], they recently determined a 1.3 Å resolution crystal structure of HRasWT−GppNHp (PDB ID 3K8Y).175 Backbone superimposition between the two structures of H-RasWT−GppNHp− Ca(OAc)2 and H-RasWT−GppNHp−CaCl2 shows that the overall structures of the two crystals are similar, yet some regions in the structure of H-RasWT−GppNHp−Ca(OAc)2 are markedly distinct from those in the structure of H-RasWT− GppNHp−CaCl2. These regions include loop L7, helix α3, and switch II (Figure 26A). In particular, the switch II region is ordered in the presence of Ca(OAc)2, whereas it is invisible or disordered in the presence of CaCl2. Inspection of the HRasWT−GppNHp−Ca(OAc)2 structure shows that Ca(OAc)2 is located among helix α3, helix α4, and loop L7 which are remote from the catalytic center (Figure 26B). Ca2+ ion in the binding site is hepta-coordinated by two oxygen atoms of

9. CONFORMATIONAL EQUILIBRIUM BETWEEN THE ON AND OFF STATES OF RAS−GTP The static snapshots of crystal structures of H-Ras−GppNHp− Ca(OAc)2 and H-Ras−GppNHp−CaCl2 emphasize that Ca(OAc)2 binding at the allosteric site induces a disorder to order transition in the switch II region with alignment of catalytic residue Q61 for catalysis (on state of the allosteric switch), while in the presence of CaCl2 the switch II region is still in a disordered conformation (off state).158,175 To further examine the dynamic conformational ensembles between the on and off states of Ras−GTP in solution, Holzapfel et al.169 recently used small molecules, dithioerythritol (DTE) (Figure 27A) and dithiothreitol (DTT) (Figure 27B), and bulk solvent composition to modulate the conformational changes of HRas−GppNHp. In the presence of 30% poly(ethylene glycol) 400 (PEG 400), CaCl2, and DTE, the authors observed a new 6629


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of H-Ras−GppNHp−Ca(OAc)2 (“ordered on” state, PDB ID 3K8Y) and H-Ras−GppNHp−CaCl2 (disordered state, PDB ID 2RGE) (Table 2). In the active site of this new ordered off conformation (PEG 400/CaCl2/DTE, PDB ID 4DLR) (Figure 27C), the catalytic residue Q61 forms a hydrogen bond with the hydroxyl group of Y32, which in turn is involved in a hydrogen bond with the γ-phosphate. Interaction of the catalytic water molecule W175 with the γ-phosphate is also observed. However, the bridging water molecule W189 observed in the ordered on state (Figure 26C) between Y32 and Q61 is missing in the ordered off state. This is attributed to the direct hydrogen-bonding interaction between Q61 and Y32 leaving no room for accommodating the bridging water molecule W189. The binding site of DTE is located between helix 3 and the switch II region (Figure 27D), which is near the active site of H-Ras−GppNHp. The DTE molecule engages in a hydrogen bond with R68 through one of its sulfur atoms and indirectly interacts with Q61 through hydrogen-bonding interactions mediated by three water molecules (W365, W308, W416) (Figure 27E). The hydroxyl group of Y96 forms two water-mediated hydrogen bonds with R68 (W345) and DTE (W365). These hydrogen-bonding interaction networks may be responsible for the ordered off state, rendering the active site in the anticatalytic conformation. Furthermore, soaking crystals in the presence of high-PEG, DTE, and 100 mM Ca(OAc)2, the authors found that DTE binds at the helix α3−switch II interface and the switch II region is in the ordered off conformation [PEG 400/Ca(OAc)2, PDB ID 3V4F in Table 2] (Figure 27F). These results suggest that under the high-PEG conditions with DTE bound near the active site, Ca(OAc)2 is unable to induce an allosteric switch of H-Ras−GTP to the ordered on state. Next, under the lower-PEG conditions (30% PEG 3350), the authors conducted two sets of soaking experiments, one beginning from the off state (set 1) and the other from the on state (set 2) to investigate the conformational transition between the two states of H-Ras−GppNHp induced by Ca(OAc)2 and DTE or DTT (Table 2). Set 1 was performed beginning with crystals containing H-Ras−GppNHp in the ordered off state in the presence of CaCl2. Addition of CaCl2 but not DTE or DTT to set 1 shows that the active site exists in two states, on (Figure 28A) and off (Figure 28B) (set 1, CaCl2 “mixed”, PDB ID 4DLS). This indicates that in the absence of CaCl2 and DTE or DTT, H-Ras−GTP is in conformational equilibrium between the two states. Addition of either 100 mM

Figure 27. Chemical structures of DTE (A) and DTT (B). (C) The structural feature of the active site of H-Ras−GppNHp in the presence of PEG 400/CaCl2/DTE (ordered off, PDB ID 4DLR). (D) Surface representation of the binding site of DTE at the helix α3−switch II interface in the ordered off conformation. (E) Water-mediated hydrogen-bonding interactions from DTE to Q61. (F) The structural feature of the active site of H-Ras−GppNHp in the presence of PEG 400/Ca(OAc)2/DTE (ordered off, PDB ID 3V4F). Green dotted lines indicate hydrogen bonds. The water molecules are depicted by red spheres. The ordered off conformation possesses the direct hydrogenbonding interaction between Q61 and Y32.

conformation of the switch II region, named “the ordered off conformation”, which is different from that observed in crystals

Table 2. Structural Features of H-Ras−GppNHp in Complex with Ca(OAc)2/CaCl2 or the Small Molecules DTE/DTT

set 1a

set 2b

PDB ID

structure

small molecules or Ions

4DLR 3V4F 4DLS 4DLX 4DLY 4DLU 4DLT 4DLV 4DLW 4DLZ

PEG 400/CaCl2/DTE ordered off PEG 400/Ca(OAc)2/DTE ordered off CaCl2 mixed CaCl2/DTE ordered off CaCl2/DTT ordered off Ca(OAc)2 ordered on Ca(OAc)2 ordered on CaCl2/DTT ordered off Ca(OAc)2/DTT ordered on Ca(OAc)2/DTE ordered off

DTE in helix α3/switch II pocket DTE in helix α3/switch II pocket none DTE in helix α3/switch II pocket DTT in helix α3/switch II pocket Ca(OAc)2 in the allosteric site Ca(OAc)2 in the allosteric site DTT in helix α3/switch II pocket Ca(OAc)2 in the allosteric site DTE in helix α3/switch II pocket

a

The set 1 experiments were performed beginning with crystals containing H-Ras−GppNHp in the ordered off state with the growth condition of CaCl2 and DTE and presoak condition of 100 mM CaCl2. bThe set 2 experiments were performed beginning with crystals containing H-Ras− GppNHp in the ordered on state with the growth condition of Ca(OAc)2 and DTE and presoak condition of 100 mM Ca(OAc)2. 6630


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Figure 28. Structural features of the active site of H-Ras−GppNHp in the presence of CaCl2 [“mixed”, ordered on (A) and ordered off (B); PDB ID 4DLS], CaCl2/DTE (ordered off, PDB ID 4DLX) (C), CaCl2/DTT (ordered off, PDB ID 4DLY) (D), and Ca(OAc)2 (ordered on, PDB ID 4DLU) (E). Green dotted lines indicate hydrogen bonds. The catalytic water molecule W175 that hydrogen bonds to the backbone groups of T35 and Q61 is depicted by a red sphere. The ordered off conformation possess the direct hydrogen-bonding interaction between Q61 and Y32. The ordered on conformation in panel E has the hydrogen-bonding interactions between Y32 and Q61 mediated by bridging water molecule W189. However, in panel A the electron density of W189 is invisible.

the pocket between helix α3 and switch II. Taken together, these data suggest that in the presence of a moderate amount of PEG the conformational equilibrium between on and off states of H-Ras−GTP is reversible and depends on the presence of small molecules that can be soaked into the crystal. However, in the presence of a high PEG concentration in the bulk solvent the equilibrium is markedly shifted toward the ordered off state.

DTE (set 1, CaCl2/DTE ordered off, PDB ID 4DLX) (Figure 28C) or 100 mM DTT (set 1, CaCl2/DTT ordered off, PDB ID 4DLY) (Figure 28D) to set 1 reveals that the active site is in the ordered off state, without the ordered on state, suggesting that DTE or DTT bound to helix α3/switch II interface shifts the conformational equilibrium of H-Ras−GTP toward the ordered off state. In sharp contrast, addition of 100 mM Ca(OAc)2 [set 1, Ca(OAc)2 ordered on, PDB ID 4DLU] (Figure 28E) to set 1 reveals that the active site is in the ordered on state, without the ordered off state, suggesting that Ca(OAc)2 bound to the allosteric site between helix α3 and loop L7 shifts the conformational equilibrium of H-Ras−GTP toward the ordered on state. In contrast, set 2 was carried out beginning with crystals containing H-Ras−GppNHp in the ordered on state in the presence of Ca(OAc)2. As expected, addition of 100 mM Ca(OAc)2 and no DTE or DTT to set 2 shows that the active site is in the ordered on state [set 2, Ca(OAc)2 ordered on, PDB ID 4DLT] (Figure 29A). Conversely, addition of 100 mM CaCl2 and 100 mM DTT to set 2 reveals that the active site is in the ordered off state (set 2, CaCl2/DTT ordered off, PDB ID 4DLV) (Figure 29B). This indicates that DTT binding shifts the conformational equilibrium of H-Ras−GTP toward the ordered off state. Interestingly, addition of 100 mM Ca(OAc)2 and 100 mM DTT to set 2 results in the structure in the ordered on state [set 2, Ca(OAc)2/DTT ordered on, PDB ID 4DLW] (Figure 29C). In this solved structure, Ca(OAc)2 is clearly bound in the allosteric site lined by helix α3 and loop L7, whereas no DTT is bound in the pocket between helix α3 and switch II. In contrast, addition of 100 mM Ca(OAc)2 and 100 mM DTE to set 2 leads to the structure in the ordered off state [set 2, Ca(OAc)2/DTE ordered off, PDB ID 4DLZ] (Figure 29D). In this solved structure, no bound Ca(OAc)2 is detected in the allosteric site, whereas DTE is clearly bound in

10. REGULATION OF RAS 10.1. Activation of Ras by SOS

Ras-specific nucleotide exchange factor SOS catalyzes the ratelimiting step in the activation of Ras by exchanging GDP for GTP.242−244 Indeed, SOS accelerates the exchange reaction by several orders of magnitude.27 Association of Ras−GDP with SOS promotes GDP release, allowing nucleotide-free Ras to bind more abundant cellular GTP.245 Human SOS1 is a large multiprotein consisting of ∼1330 residues246 (Figure 30A). It includes an N-terminal domain (residues 1−198), a Dbl homology (DH) domain (residues 199−404), a pleckstrin homology (PH) domain (residues 405−550), a core catalytic domain (SOScat) (residues 551−1050) that comprises the Ras exchanger motif (Rem) domain (residues 551−750) and Cdc25 domain (residues 751−1050), and a C-terminal region (residues 1051−1333) that provides docking sites for the adaptor protein Grb2, which recruits SOS to the membrane upon receptor activation.246 To date, a wealth of crystal structures of H-Ras−SOS complexes have been determined with different constructs of SOS domains and nucleotide-bound Ras states, including SOScat:H-Ras nucleotide-free complex (hereafter referred to as SOScat:H-Ras), SOSDH‑PH‑cat, H-Ras− GppNHp:SOSDH‑PH‑cat:H-Ras, H-Ras−GDP·Pi:SOSDH‑PH‑cat:HRas, and H-Ras−GppNHp:SOScat:H-Ras:compounds, which 6631


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conformational changes of the switch I and switch II regions.247 Comparison of the structure of nucleotide-free H-Ras in the presence of SOScat to that of H-Ras−GppNHp (PDB ID 5P21) exhibits that insertion of helix αH of Cdc25 into the nucleotidefree H-Ras active site results in residue L938 preventing Mg2+ binding and residue E942 overlapping the binding site of the αphosphate of the nucleotide, but not the base or the ribose binding sites of the nucleotide (Figure 30F). On the basis of this observation, a mechanism of dissociation or association of a nucleotide to the H-Ras active site triggered by SOS was proposed to be as follows:248 in the dissociation process the phosphate moieties of GDP are released first after binding of SOS, and then the base and ribose moieties of GDP are released. In contrast, in the association process, the base and ribose moieties of GTP bind first to the nucleotide-binding site of H-Ras. Significant conformational changes of the switch I and switch II regions would occur to rebuild the binding sites for phosphate and Mg2+ and subsequently displace SOS. In 2003, Margarit et al.249 determined the crystal structure of SOScat in complex with H-RasA59G, a mutation at the beginning of the switch II region that demolishes both GTP hydrolysis and nucleotide exchange. The solved structure (PDB ID 1NVU) shows that the nucleotide-free H-RasA59G is located in the active site from the Cdc25 domain of SOScat (Figure 31A), which is very similar to the position of nucleotide-free H-RasWT in the previously reported SOScat:H-Ras complex. Unexpectedly, in the structure of mutant SOScat:H-RasA59G complex, another H-RasA59G complexed with GTP is bound to the new binding site between the Rem and the Cdc25 domains of SOScat (Figure 31A), which is distal to the active site of nucleotide-free H-RasA59G. Thus, the resulting mutant structure is a ternary complex, including two molecules of H-RasA59G and one molecule of SOScat (hereafter referred to as H-RasA59G− GTP:SOScat:H-RasA59G). In a similar vein, using H-RasWT complexed with GppNHp, the authors also obtained the ternary complexes (H-RasWT−GppNHp:SOScat:H-RasWT, PDB ID 1NVW) (Figure 31B) with the same positions of H-RasWT and H-RasA59G in both the active and distal sites of the ternary complexes. Subsequently, they also determined the crystal structure of SOScat complexed with both H-RasWT and HRasY64A, with the Y64A mutation locating on the switch II region that disrupts the binding of H-RasY64A to the active site of SOS.250 The obtained ternary complex (PDB ID 1NVV) (Figure 31C) clearly shows that H-RasY64A−GppNHp and HRasWT are bound to the distal and active sites of SOScat (HRasY64A−GppNHp:SOScat:H-RasWT), respectively, which are essentially the same with H-RasA59G and H-RasWT in the two sites observed in the respective structures of H-RasA59G− GTP:SOScat:H-RasA59G and H-RasWT−GppNHp:SOScat:HRas WT . Comparison of the structure of H-Ras Y64A − GppNHp:SOScat:H-RasWT to that of SOScat:H-RasWT clarifies that binding of H-RasY64A−GppNHp at the distal binding site between the Rem and Cdc25 domains of SOScat has a profound influence on the conformational change of the Rem domain, resulting in the rotation of the Rem domain relative to the Cdc25 domain by ∼10° (Figure 31D). The rotation of the Rem domain triggered by H-RasY64A−GppNHp binding in turn echoes the interactions between the helical hairpin of SOScat and the switch I region of nucleotide-free H-RasWT in the active site. For example, compared with no H-Ras bound to the distal site (Figure 31D), in the presence of H-RasY64A−GppNHp at the distal site, residues D30 and E31 at the active site on the switch I region of the nucleotide-free H-RasWT engage in polar

Figure 29. Structural features of the active site of H-Ras−GppNHp in the presence of Ca(OAc)2 (ordered on, PDB ID 4DLT) (A), CaCl2/ DTT (ordered off, PDB ID 4DLV) (B), Ca(OAc)2/DTT (ordered on, PDB ID 4DLW) (C), and Ca(OAc)2/DTE (ordered off, PDB ID 4DLZ) (D). Green dotted lines indicate hydrogen bonds. The catalytic water molecule W175 that hydrogen bonds to the backbone groups of T35 and Q61 is depicted by a red sphere. The ordered off conformation possess the direct hydrogen-bonding interaction between Q61 and Y32. The ordered on conformation has no direct interaction between Q61 and Y32. The space between them is enough to accommodate the bridging water molecule W189, but the electron density of W189 is invisible.

provide comprehensive information on the mechanism of activation of Ras by SOS. In the first crystal structure of H-Ras−SOS complex, solved in 1998, the catalytic domain of SOS (residues 568−1044) was bound to the nucleotide-free H-Ras catalytic domain (SOScat:H-Ras, PDB ID 1BKD).193 The determination of the crystal structure of SOScat:H-Ras shows that H-Ras is bound to the Cdc25 domain (residues 752−1044) of SOScat and has no direct contacts with the Rem domain (residues 568−741) of SOScat (Figure 30B). The regions of H-Ras that interact with the Cdc25 domain of SOScat are derived primarily from the Ploop, helix α1, and the switch I and switch II regions (Figure 30C). The most significant conformational change of H-Ras in response to SOScat binding results from the insertion of the helical hairpin from helices αH and αI of Cdc25 (Figure 30D). The switch I and switch II regions of H-Ras undergo a marked deviation, leading to the opening of the nucleotide binding site (Figure 30E). The opening would weaken the binding affinity of the nucleotide to H-Ras, facilitating the release of the nucleotide and the subsequent rebinding of a nucleotide to the H-Ras active site. The position of helix αH of Cdc25 is located between the switch I and switch II regions, which suggests that helix αH of Cdc25 plays a pivotal role in controlling the nucleotide dissociation and association through fine-tuning the 6632


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Figure 30. (A) Schematic diagram showing the domain organization of human SOS1. (B) Surface representation of the crystal structure (SOScat:HRasWT, PDB ID 1BKD) of nucleotide-free H-Ras (orange) in complex with the catalytic domain of SOS (SOScat) consisting of the Rem (pink) and the Cdc25 (cyan) domains. (C) The interaction between H-Ras depicted as a cartoon and Cdc25 domain depicted as a surface. (D) Cartoon representation of the interaction between H-Ras and Cdc25 domain. The helical hairpin from the helices αH and αI of Cdc25 domain is colored red. (E) Surface representation of the nucleotide-free H-Ras. (F) Backbone superimposition of the crystal structure of H-Ras (nucleotide free)−SOScat (orange) onto that of H-RasWT−GppNHp (PDB ID 5P21, green). The helix αH is colored red. Residues L938 and E942 and GppNHp are depicted by stick models. Mg2+ ion is depicted by a green sphere. Reproduced with permission from ref 54. Copyright 2015 Wiley Press.

Figure 31. Surface representations of the crystal structures of the ternary complexes H-RasA59G−GTP:SOScat:H-RasA59G (PDB ID 1NVU) (A), HRasWT−GppNHp:SOScat:H-RasWT (PDB ID 1NVW) (B), and H-RasY64A−GppNHp:SOScat:H-RasWT (PDB ID 1NVV) (C). Nucleotide-free H-Ras in the active site of SOScat is in orange and GTP-/GppNHp-bound H-Ras in the distal allosteric site of SOScat is in yellow. The Rem and the Cdc25 domains of SOScat are in pink and cyan, respectively. (D) Middle: cartoon representation of the backbone superimposition of crystal structure of SOScat:H-RasWT (PDB ID 1BKD, pink) onto that of H-RasY64A−GppNHp:SOScat:H-RasWT (cyan). Changes of the Rem domain of SOScat are shown in the expanded illustration (middle, right). Changes in the switch I region of nucleotide-free H-Ras at the active site of SOScat are shown in the expanded illustration (middle, left). Reproduced with permission from ref 54. Copyright 2015 Wiley Press.

interactions with residues K602 and R950 of SOScat and residues K963 and S959 of SOScat, respectively. These observations suggest that binding of H-RasY64A−GppNHp at the distal site allosterically enhances the interactions between the switch I region of nucleotide-free H-RasWT at the active site and SOScat, which may in turn promote the nucleotide exchange rate. To test this hypothesis, experimental characterization of SOS-catalyzed nucleotide exchange was performed

through the measurement of nucleotide release rates from HRas using fluorescence spectroscopy. The results exhibited that the GTP-loaded forms of H-RasWT, H-RasA59G, and H-RasY64A markedly increase the rate of SOScat-stimulated mantGDP (an N-methylanthranyl derivative of GDP) release from H-Ras compared to the absence of the GTP-loaded form of H-RasWT. In striking contrast, GDP-loaded forms of H-RasWT, H-RasA59G, and H-RasY64A have subtle effects on the rate of SOScat6633


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Figure 32. (A) Surface representation of the crystal structure of SOSDH‑PH‑cat (PDB ID 1XD4). The DH and PH domains are in blue and green, respectively. The Rem and the Cdc25 domains of SOScat are in pink and cyan, respectively. (B) Cartoon representation of the backbone superimposition of the crystal structure of SOSDH‑PH‑cat (cyan) onto that of H-RasY64A−GppNHp:SOScat:H-RasWT (PDB ID 1NVV, pink). The distal allosteric H-RasY64A−GppNHp in the ternary complex overlaps with the DH domain of SOSDH‑PH‑cat. (C) Surface representation of the crystal structure of H-RasY64A−GDP-Pi:SOScat:H-RasWT (PDB ID (PDB ID 1XD2). (D) Cartoon representation of the backbone superimposition of crystal structure of H-RasY64A−GppNHp:SOScat:H-RasWT (pink) onto that of H-RasY64A−GDP-Pi:SOScat:H-RasWT (cyan).

location of the DH domain in the SOSDH‑PH‑cat structure prevents H-RasY64A−GppNHp from binding to the distal allosteric binding site of SOScat (Figure 32B). This observation suggests that H-Ras−GTP is incapable of stimulating the nucleotide exchange activity of SOSDH‑PH‑cat, because the existence of the DH domain would impede the access of HRas−GTP to the distal binding site of SOScat. Experimental characterization of SOS-catalyzed nucleotide exchange was performed using fluorescence spectroscopy to probe the effects of the DH-PH domains on the nucleotide exchange activity of SOS. The results revealed that compared with SOScat, the release of GDP from H-Ras is approximately 3 times slower in the presence of SOSDH‑PH‑cat. Remarkably, the addition of HRasY64A−GppNHp significantly enhances the release of GDP from H-Ras by SOScat. In sharp contrast, no significant increase in the rate of GDP release was observed in SOSDH‑PH‑cat. The triple mutant (E268A/M269A/D271A) in the DH domain of SOSDH‑PH‑cat (hereafter referred to as SOSDH‑PH‑cat:triple.mut) was subsequently constructed to attenuate the inhibitory effect of the DH domain on the rate of SOSDH‑PH‑cat-catalyzed nucleotide exchange in H-Ras−GppNHp, because the triple mutations on the DH domain would weaken the interaction between the DH and Rem domains. The results showed that the rate of GDP release in SOSDH‑PH‑cat:triple.mut in the presence of H-RasY64A−GppNHp (a 1:1 ratio of H-RasY64A−GppNHp and SOSDH‑PH‑cat:triple.mut) is comparable to that of SOScat in the absence of H-RasY64A−GppNHp. Especially, when the ratio of H-RasY64A−GppNHp and SOSDH‑PH‑cat:triple.mut increases to 20:1, the rate of GDP release in the SOSDH‑PH‑cat:triple.mut in the presence of H-RasY64A−GppNHp is similar to that of SOScat in the presence of H-RasY64A−GppNHp. Addition of H-RasY64A− GDP to SOSDH‑PH‑cat:triple.mut can also moderately increase the

stimulated mantGDP release. Taken together, these data indicate the existence of a positive feedback mechanism in the activation of Ras by SOS. Furthermore, Boykevisch et al.251 found that the positive feedback loop between H-Ras−GTP and SOS results in an increase in the amplitude and duration of Ras activation after the stimulation of EGF, which in turn leads to an increase in the activity of downstream proteins of the pathway, as characterized by sustained EGF-induced ERK phosphorylation and enhanced serum response element (SRE)dependent transcription. These data suggest that Ras signaling is dynamically regulated by SOS-mediated positive feedback.252 On the basis of theoretical analyses and stochastic computer simulations to investigate how activation of Ras by SOS is regulated in lymphoid cells, Das et al.253 also pinpointed that a positive feedback loop is associated with SOS-mediated Ras activation. Recently, Iversen et al.254 developed a singlemolecule enzymatic assay that enables detailed observation of the activation of membrane-linked H-Ras by SOS. They further uncovered that the positive feedback activation of H-Ras by SOS is through a minority of highly active states of SOS, rather than the ensemble average of SOS.254 In 2004, Sondermann et al.255 determined the crystal structure of SOSDH‑PH‑cat that includes the DH−PH domains in addition to the catalytic domain (SOScat). The SOSDH‑PH domains precede the SOScat domain (Figure 30A). The 3.62 Å resolution crystal structure of SOSDH‑PH‑cat (PDB ID 1XD4) shows that the PH domain associates exclusively with the DH domain, which in turn packs against the face of the Rem domain (Figure 32A). The DH domain is distal to the Cdc25 domain, and no direct contact between the DH and Cdc25 domains exists. Comparison of the structure of SOSDH‑PH‑cat to that of H-RasY64A−GppNHp:SOScat:H-RasWT shows that the 6634


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Figure 33. Chemical structures of aminopiperidine indole compounds 1−4 that increase SOS-catalyzed nucleotide exchange.

change, suggesting that 4-mediated activation does not need Ras binding to the allosteric site. In further support of this notion, biochemical experiments revealed that compound 4 is also able to activate nucleotide exchange in the presence of autoinhibited SOSDH‑PH‑cat. Collectively, these data suggest that the activation of nucleotide exchange by compound 4 is through a mechanism distinct from the previously reported positive feedback mechanism that requires binding of Ras− GTP to the allosteric site of SOS. The determination of X-ray crystal structures of H-RasY64A−GppNHp:SOScat:H-RasWT in complex with compounds 1 (Figure 34A, PDB ID 4NYJ), 2 (Figure 34B, PDB ID 4NYM), and 3 (Figure 34C, PDB ID 4NYI) clarified that the three compounds are located on the Cdc25 domain of SOScat in the vicinity of the switch II region of Ras at the active site of SOScat (due to limited water solubility, compound 4 was resistant to crystallization). Backbone superimposition of the structure of nucleotide-free H-Ras onto those of GppNHp-bound and GDP-bound H-Ras shows that compared with the GppNHp-/GDP-bound H-Ras, the switch I and switch II regions of nucleotide-free H-Ras undergo the most remarkable structural changes (Figure 34D). Moreover, the switch I is completely removed from the nucleotide-binding site. In addition, the P-loop and the L8 loop that connets β5 to helix α4 also show apparent structural changes in the nucleotide-free H-Ras. Mutations of F890, L901, and H905 within the hydrophobic binding site of the compounds on the Cdc25 domain of SOScat to alanine eradicated the compound-induced activation of mediated SOScat−nucleotide exchange, revealing the importance of this binding site in the regulation of the activation of Ras by SOS. Cell-based experiments showed that the levels of H-Ras−GTP increase in response to the addition of compounds 2 and 4 to HeLa cells, which is in good agreement with the increase in nucleotide exchange activity in vitro. Furthermore, the ability of compound 4 to affect cell growth and transformation showed that H-Ras-harboring cancer cells weaken cell proliferation and anchorage-independent growth in response to compound 4. This observation suggests that this pocket on SOS provides a valuable new avenue for the discovery of potent inhibitors of Ras signaling. On the basis of Ras−SOS protein−protein interactions, it is feasible to design small molecules or peptides bound to the Ras−SOS interface, thereby resulting in inhibiting SOScatalyzed Ras activation, which represents an attractive therapeutic strategy for the treatment of Ras-driven cancers.

rate of GDP release from H-Ras. However, the rate of GDP release in the presence of SOSDH‑PH‑cat:triple.mutwhich is accelerated by H-RasY64A−GDPis markedly slower than that by H-RasY64A−GppNHp, but it is faster than that in the presence of SOSDH‑PH‑cat induced by H-RasY64A−GppNHp. The measurement of binding affinities of H-RasY64A−GppNHp and H-RasY64A−GDP to the distal binding site of SOScat shows that H-RasY64A−GppNHp (Kd ∼ 3.6 μM) binds to the distal binding site with a higher affinity than H-RasY64A−GDP (Kd ∼ 24.5 μM). The determined crystal structure of H-RasY64A− GDP:SOScat:H-RasWT complex (PDB ID 1XD2) shows that this structure is similar to that of H-RasY64A−GTP:SOScat:HRasWT complex, with the binding of GDP and a phosphate ion (Pi) to the distal allosteric binding site of SOScat (Figure 32C,D). Cumulatively, these data suggest that the DH−PH domains play a role in the autoinhibition of SOS through blocking a distal allosteric binding site for Ras.256 However, how the autoinhibition of SOS is relieved by decoupling the DH−PH domains from the Rem domain in vivo is still unresolved. A possible explanation may be that recruitment of SOS to the PM causes conformational changes that mitigate the inhibitory effect of DH−PH domains and allow Ras−GDP to bind to SOS’s allosteric site.257 In fact, membrane PIP2 binding to SOS’s PH domain increases SOS catalytic activity.258 In addition, a construct of SOScat without the DH−PH domains leads to Ras signaling without the requirement of membrane targeting or external stimulus.259 Recently, Burns et al.260 identified a small molecule, a 3-(4aminopiperidinyl)methylindole attached to glycine (compound 1) (Figure 33), that weakly enhanced SOScat-catalyzed nucleotide exchange in vitro. Structure−activity modifications of compound 1 yielded more potent compounds 2−4 (Figure 33) compared to compound 1 in the activation of nucleotide exchange. Compound 4 is the most potent small molecule, with an EC50 of 14 μM. Nucleotide-exchange assays showed that compound 4 increases SOScat-catalyzed nucleotide exchange, but it has no effect on the intrinsic nucleotide exchange on HRas without the stimulation of SOScat. This indicates that compound 4 activates nucleotide exchange in a SOS-dependent pattern. Furthermore, addition of 100 μM of compound 4 to either SOScat‑W729E or SOScat‑L687E/R688A enabled activation of nucleotide exchange. The two constructs of SOScat mutants are resistant to binding of Ras−GTP to the distal allosteric binding site of SOS. As of now, compound 4 can efficiently increase SOScat‑W729E- and SOScat‑L687E/R688A-mediated nucleotide ex6635


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magnitude with 19 s−1 (measured at 25 °C),271 thereby terminating and inactivating Ras signaling. The determination of the crystal structure of the complex between H-Ras complexed with GDP, and the GTPase-activating domain (residues 718−1037) of the human Ras-specific GAP, p120GAP (hereafter referred to as GAP), in the presence of AlF3 (PDB ID 1WQ1)272 showed that H-Ras interacts mainly with GAP through the P-loop and the switch I and switch II regions (Figure 36).273−275 Remarkably, detailed analysis of the H-Ras active site shows that the positively charged residue R789 of GAP, the so-called arginine finger, interacts with the αand β-phosphate groups of GDP and AlF3 (Figure 36). This observation suggests that the arginine finger R789 of GAP protrudes into the active site of H-Ras to neutralize developing negative charges in the transition state.276−280 In addition, the engagement of GAP with the switch II region of H-Ras may restrict the flexibility of the switch II region, which stabilizes the intrinsically mobile H-Ras to correctly position the catalytic residue Q61.209,281,282 Indeed, in this structure, the side chain carbonyl and amine groups of Q61 form a hydrogen bond with the catalytic water molecule and the backbone carbonyl group of arginine finger R789, respectively. These interactions enable the Q61 to extract a hydrogen atom from the catalytic water and subsequently the negative hydroxyl ion can attack the γphosphorus of GTP to perform GAP-mediated GTP hydrolysis.145,283−288 Biochemical experiments have established that mutation of Q61 impairs the GAP-mediated GTP hydrolysis as a consequence of the disruption of the interaction between Q61 and the catalytic water molecule.212−215 Recent MD simulations of K-Ras4BWT−GTP have revealed that K-Ras4BWT−GTP in solution exists in the active and inactive states. To further probe the effect of GAP on the conformational dynamics of K-Ras4BWT−GTP, simulations of K-Ras4BWT−GTP−GAP complex were performed on the basis of the initial crystal structure of H-RasWT−GDP−AlF3−GAP complex in which H-Ras was mutated to K-Ras4B and the GDP and AlF3 were substituted by GTP to model the K-Ras4BWT− GTP−GAP complex. The calculations of the probability distributions representing the PMF for the atom-pair distances, d1 (defined by the distance from the Cα atom of switch II residue G60 to the Pβ atom of GTP) and d2 (defined by the distance from the Cα atom of switch I residue T35 to the Pβ atom of GTP), showed that K-Ras4BWT−GTP exhibits one energy-minima basin in the presence of GAP (Figure 37A), with the d1 and d2 values ranging from 6.5 to 7.8 and 6.0 to 6.5 Å, respectively, which represent the active state. These data suggest that compared to the free K-Ras4BWT−GTP, binding of GAP to K-Ras4BWT−GTP shifts the population of KRas4BWT−GTP to the active state. In the active site of KRas4B, G13 partakes in a hydrogen bond with the β−γ bridging oxygen atom of GTP, Q61 interacts with the catalytic water and forms a hydrogen bond with the γ-phosphate of GTP, and GAP provides the arginine finger R789 that protrudes into the active site of K-Ras4B by virtue of salt bridge interactions with the αand γ-phosphates of GTP. These interaction patterns between K-Ras4BWT−GTP and GAP are consistent with the results of previous MD simulations of H-RasWT−GTP−GAP complex on very short time scales (∼1 ns), which also revealed that Q61 of H-Ras and R789 of GAP played a pivotal role in GTP hydrolysis.209,289 MD simulations of oncogenic mutations (G12C, G12D, G12V, G13D, and Q61H) in the K-Ras4B−GTP−GAP complex were deployed to explore how oncogenic mutations

Figure 34. Surface representations of X-ray cocrystal structures of compounds 1 (PDB ID 4NYJ) (A), 2 (PDB ID 4NYM) (B), and 3 (PDB ID 4NYI) (C) bound to the H-RasY64A−GppNHp:SOScat:HRasWT ternary complex. The catalytic nucleotide-free H-RasWT and the allosteric H-RasY64A−GppNHp are in orange and yellow, respectively. The Rem and the Cdc25 domains of SOScat are in pink and cyan, respectively. A close-up view of the hydrophobic pocket of the compounds between the switch II region (blue) of the catalytic HRasWT and the Cdc25 domain of SOScat is shown on the left. Residues F890, L901, and H905 within the hydrophobic pocket are in red. (D) Cartoon representation of the structure of nucleotide-free H-Ras (blue) superimposed onto that of GppNHp-bound H-Ras (pink) and GDP-bound H-Ras (cyan).

Currently, a wide range of small molecules or peptides have been reported to inhibit the SOS-catalyzed Ras activation (Figure 35), including indoles (1),261 phenols (2),261 sulfonamides (3),261 BZIM (4),262 INDL (5),262 DCAI (6),262 bisphenol A (7),263 NSC-658497 (8),264 UC-773587 (9),265 UC-857993 (10),265 compounds 1−3 (11−13),266 andrographolide (AGP) (14),267 sugar-derived compounds (15),268 stabilized α-helices of SOS1 (SAH-SOS1) peptides,269 and hydrogen-bond surrogate (HBS) α-helix 3 (HBS 3, a SOS1 αH mimetic).270 10.2. Inactivation of Ras by GAP

Ras has a very slow intrinsic rate of GTP hydrolysis of 4.7 × 10−4 s−1 at 37 °C.248 Significantly, Ras-specific GAPs accelerate the intrinsic GTP hydrolysis rate of Ras by 5 orders of 6636


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Figure 35. Chemical structures of small molecule compounds 1−15 that inhibit SOS-catalyzed Ras activation.

Figure 36. Complex (PDB ID 1WQ1) between H-Ras depicted by a cartoon in complex with GDP and AlF3 and the catalytic domain of GAP depicted by a surface. A close-up view of the active site is shown on the left. The catalytic residue Q61 of H-Ras, the arginine finger R789 of GAP, GDP, and AlF3 are depicted by stick models. The catalytic water molecule and Mg2+ ion are depicted by red and green spheres, respectively. Hydrogen bonds are depicted by green dotted lines.

impair the GAP-mediated GTP hydrolysis. The simulations indicated that the oncogenic mutations disturb the “correct” arrangements of the catalytic residue Q61 of K-Ras4B and the arginine finger of R789 of GAP in the active site for catalysis,

thereby leading to the mutant K-Ras4B resistance to GAPmediated GTP hydrolysis.290 As a result, mutant K-Ras4B proteins persist in the active GTP-bound form that can interact 6637


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analyses showed that only mutations such as E31, T35, and E37 in the switch I region of H-Ras significantly impair the association of RafRBD to mutant H-Ras, suggesting that the switch I region of H-Ras directly interacts with RafRBD.302 Unexpectedly, another mutation on the CRD of C-Raf (residues 139−184) (hereafter referred to as RafCRD), C168S, impaired the interaction of mutant Raf with H-Ras,303 indicating that in addition to engagement with RafRBD, Ras may also interact with RafCRD. Direct measurement of Ras binding to individual RafRBD and RafCRD in vitro showed that mutations of T35A and E37G in the switch I region of HRas impair the interaction between H-Ras and RafRBD, but they have no effect on the association between H-Ras and RafCRD.304 Conversely, mutations of G60A and Y64W in the switch II region of H-Ras impair the interaction between H-Ras and RafCRD, but they have no effect on the association between H-Ras and RafRBD.304 These observations suggest that RafRBD and RafCRD interact with the switch I and switch II regions of H-Ras, respectively. A binding affinity assay reveals that RafRBD binds to H-Ras with an affinity of 18 nM, while RafCRD binds with a much lower affinity of 20 μM.194 Mutations on the switch I region of H-Ras have previously been well-established to abolish oncogenic H-Ras transforming activity.305,306 Even though mutations on the switch II region do not impair RafRBD binding to H-Ras, the G60A and Y64W mutations eliminate oncogenic H-Ras transforming activity. This indicates that Ras interaction with RafCRD is required for the activation of Raf.307,308 Therefore, RafRBD and RafCRD may be orchestrated in promoting the association with the PM in vivo.309 However, the mechanism by which the association of RafRBD and RafCRD with Ras contributes to the activation of the Raf kinase domain is currently unresolved. To determine the structural basis of how Ras interacts with Raf, in 1996 Nassar et al.,310 solved a 2.0 Å resolution crystal structure of the complex between the H-Ras homologue Rap1A bound to GppNHp and RafRBD (PDB ID 1GUA). Rap1A is highly homologous to Ras with 57% sequence identify and shares almost the same effector binding interface as Ras. Double mutations of E30D and K31E on the switch I region of Rap1A were performed to mimic the corresponding switch I region of Ras. The 3D structure of the Rap1AE30D/K31E− GppNHp−RafRBD complex reveals that the RafRBD interacts mainly with the switch I region of Rap1AE30D/K31E (Figure 38A), in good agreement with previous experiments indicating that mutations on the switch I region of H-Ras obviate the interaction between H-Ras and RafRBD.305,306 The binding interface between Rap1A and RafRBD derives mainly from the strand β2 of the RafRBD and the strand β2 and the switch I region of the Rap1AE30D/K31E. Detailed analysis of the interaction between Rap1AE30D/K31E and RafRBD exhibits that contacts on Rap1AE30D/K31E stem from residues E31, D33, E37, and D38, with strong/weak salt bridges forming among E31 Rap1A −K84 Raf , D33 Rap1A −K84 Raf , D33 Rap1A −R73 Raf , E37Rap1A−R59Raf, E37Rap1A−R67Raf, and D38Rap1A−R89Raf (Figure 38B). A series of hydrogen bonds also exists between the backbone groups of the strand β2 from both RafRBD and Rap1AE30D/K31E. Importantly, the strong and weak salt bridges between residues E31 and D33 of Rap1AE30D/K31E and residue K84 of RafRBD in Rap1AE30D/K31E−RafRBD complex were not observed in the previous Rap1AWT−RafRBD complex.311 However, comparison of NMR CSPs between Rap1AE30D/K31E−RafRBD and Rap1AWT−RafRBD complexes showed that E31 in Rap1AE30D/K31E may not become the

Figure 37. Probability distributions for two atom-pair distances, d1 (defined by the distance from the G60 Cα atom to the GDP Pβ atom) and d2 (defined by the distance from the T35 Cα atom to the GDP Pβ atom), were calculated on the MD snapshots of K-Ras4BWT−GTP− GAP and converted into the surface plot representing the potential of mean force.

with its downstream effector, such as Raf, leading to a sustained oncogenic signal.190

11. ASSOCIATION OF RAS−GTP WITH EFFECTOR PROTEINS In the active GTP-bound state, Ras can associate with its downstream effectors, referred to as “Ras effectors”,291−293 including Raf kinase, PI3K, RalGDS, and NORE, thereby activating several signal transduction pathways in the cell. All reported Ras effectors contain a conserved structural domain referred to as the RBD, which binds to Ras in a nucleotidedependent manner.294 Ras−GTP strongly interacts with RBD with dissociation constants (Kd) in the range of 0.01−3 μM.233 In contrast, in the inactive GDP-bound state, Ras weakly interacts with RBD with Kd values in the upper micromolar range, which decreases by approximately 1000-fold compared with the binding affinities of Ras−GTP to Ras effectors.295 In this context, Ras−GDP loses its ability to interact with Ras effectors. To date, several crystal structures of Ras in complex with RBD of Ras effectors in the GppNHp-bound states have been solved, providing a detailed insight into the interactions between Ras and RBD.296 11. 1. Interaction with Raf

Mammals express three Raf paralogs (A-Raf, B-Raf, and C-Raf), which participate in the Ras−Raf−MEK−ERK signal transduction cascade.297 Each of the Raf isoforms contains three conserved regions (CR):298 CR1, CR2, CR3. CR1 consists of a RBD and a cysteine-rich domain (CRD), which can bind two Zn2+ ions. CR2 is a serine/threonine-rich domain, which can bind to 14-3-3, a regulatory protein. CR3 is the kinase domain, which is located near the C-terminus. Previously, yeast two-hybrid and in vitro binding studies ascertained that the RBD (residues 55−131) of C-Raf (also known as Raf-1) (hereafter referred to as RafRBD) directly interacts with H-Ras−GTP.299−301 Furthermore, mutational 6638


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Figure 38. (A) Cartoon representation of the crystal structure (PDB ID 1GUA) of the complex between Rap1AE30D/K31E−GppNHp (pink) and RafRBD (blue). The switch I region of Rap1AE30D/K31E is in red. (B) The binding interface between Rap1AE30D/K31E−GppNHp and RafRBD. Salt bridges are represented by blue dotted lines. (C) Middle: electrostatic representation of the complex between Rap1AE30D/K31E− GppNHp and RafRBD. The components of the complex have been separated and tuned toward the reader to show an electrostatic representation of the binding interface in the RafRBD (middle, right) and Rap1AE30D/K31E−GppNHp (middle, left). Positive potential is colored blue and negative potential red.

Figure 39. (A) Cartoon representation of the backbone superimposition of crystal structure of H-RasWT−GppNHp−RafRBD (PDB ID 4G0N, pink) onto that of Rap1AE30D/K31E−GppNHp−RafRBD (PDB ID 1GUA, cyan). (B) The binding sites of Ca(OAc)2 and DTE in the structure of H-RasWT−GppNHp−RafRBD are shown. (C) The active site of H-RasWT−GppNHp-RafRBD showing Y32, Q61, and the catalytic (W175) and bridging (W189) water molecules near the nucleotide. Hydrogen bonds are depicted by green dotted lines. (D) The binding interface between H-RasWT−GppNHp and RafRBD. Salt bridges are represented by blue dotted lines.

major salt bridge partner of K84 in RafRBD.312 Subsequent MD simulations of a model structure of H-Ras−GTP−RafRBD and a mutational experiment of D33A H-Ras suggested that D33 in H-Ras is the major salt bridge partner with K84 in RafRBD. Overall, the interaction of Rap1AE30D/K31E with RafRBD demonstrates that the formation of the complex derives mainly from the negatively charged residues of Rap1AE30D/K31E and the positively charged residues of RafRBD. Analysis of the electrostatic surface on the binding interface also confirms this hypothesis (Figure 38C), which shows that the binding interface on Rap1AE30D/K31E and RafRBD is negatively and positively charged, respectively. Recently, Fetics et al.162 solved the first crystal structure of the complex between H-RasWT−GppNHp and RafRBD (PDB ID 4G0N), together with the complex between the oncogenic H-RasQ61L mutant bound to GppNHp and RafRBD (PDB ID 4G3X). Backbone superimposition of the crystal structure of HRas WT −GppNHp−RafRBD to that of Rap1A E30D/K31E− GppNHp−RafRBD shows that the binding interface between H-RasWT−GppNHp and RafRBD as well as the conformation of the switch I region of H-Ras in the H-RasWT−GppNHp− RafRBD complex is very similar to that in the Rap1AE30D/K31E− GppNHp−RafRBD complex (Figure 39A). However, the switch II region between H-RasWT and Rap1AE30D/K31E shows some differences, with the invisible electron density for residues Y64 and S65 on the switch II region of H-RasWT. In particular, in the structure of H-RasWT−GppNHp−RafRBD, Ca(OAc)2 is bound to the allosteric site and a small molecule DTE is bound to the helix α3/switch II interface (Figure 39B). In the active site of H-Ras in the H-RasWT−GppNHp−RafRBD complex, the catalytic residue Q61 on the switch II region interacts with the bridging water molecule W189, which in turn interacts with the side chain of Y32 and γ-phosphate, and the catalytic water

W175 interacts with the γ-phosphate (Figure 39C). These observations suggest that the conformation of H-RasWT− GppNHp in complex with Ca(OAc)2 and DTE in the presence of RafRBD represents an ordered on conformation (R state).67 However, previous crystal studies showed that in the absence of RafRBD, the active site of H-RasWT−GppNHp bound to Ca(OAc)2 and DTE is in an ordered off conformation (T state).169 This difference indicates that RafRBD plays an important role in the allosteric activation of intrinsic GTP hydrolysis. Analysis of the interaction between H-RasWT− GppNHp and RafRBD reveals that both residues E31 and D33 on the switch I region of H-Ras form salt bridges with residue K84 of RafRBD, and residues E37 and D38 on the switch I region of H-Ras engage in salt bridges with residues R59 and R67, and residue R89 of RafRBD (Figure 39D), respectively. These interactions are also conserved in the structure of Rap1AE30D/K31E−GppNHp−RafRBD.310 However, one salt bridge between D33 of Rap1AE30D/K31E and R73 of RafRBD in the Rap1AE30D/K31E−GppNHp−RafRBD complex is not observed in the structure of H-RasWT−GppNHp−RafRBD complex. In the active site of H-RasQ61L in the H-RasQ61L−GppNHp− RafRBD complex (Figure 40A), the bridging water molecule W189 is missing owing to the Q61L mutation on the switch II region that leaves no room for the W189. This effect leads to the direct hydrogen-bonding interaction between Y32 and the γ-phosphate, which is a feature of the ordered off conformation (T state). In addition, the catalytic water W175 is also not observed in the active site, together with the lack of Ca(OAc)2 in the allosteric site. In the binding interface between H-RasQ61L and RafRBD (Figure 40B), the electron density for the side chain of K84 of RafRBD is invisible and only one salt bridge 6639


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Figure 40. (A) The active site of H-RasQ61L in the crystal structure of H-RasQ61L−GppNHp−RafRBD (PDB ID 4G3X). Hydrogen bonds are depicted by green dotted lines. (B) The binding interface between H-RasQ61L−GppNHp and RafRBD. Salt bridges are represented by blue dotted lines. (C) Cartoon representation of the backbone superimposition of the crystal structure of H-RasWT−GppNHp−RafRBD (PDB ID 4G0N, pink) onto that of H-RasQ61L−GppNHp−RafRBD (cyan).

between E37 of H-RasQ61L and R59 of RafRBD is observed. Compared with the formation of numerous salt bridge networks between H-RasWT and RafRBD in the H-RasWT− GppNHp−RafRBD complex (Figure 39D), mutation of Q61L on the switch II region of H-Ras has a significant effect on the binding interface between H-RasQ61H and RafRBD. Further backbone superimposition of the crystal structure of HRas Q61L −GppNHp−RafRBD onto that of H-Ras WT − GppNHp−RafRBD shows that the Q61L mutation not only affects the conformations of the switch I and switch II regions on the H-Ras but also perturbs the L4 loop on RafRBD away from the binding interface (Figure 40C). A series of MD simulations based on H-RasWT−GTP, H-RasQ61L−GTP, HRas WT −GTP−RafRBD, and H-Ras Q61L −GTP−RafRBD, coupled with the subsequent dynamical network analysis, revealed that in the absence of RafRBD, the Q61L mutation increases the conformational flexibility of the switch II region in H-RasQ61L and that in the presence of RafRBD this mutation quenches the motion of the switch II in H-RasQ61L. This effect leads to the disruption of the connectivity and thus allosteric modulation of the active site in the H-RasQ61L−GTP−RafRBD and influences the conformational dynamics of the distal L4 loop in the H-RasQ61L−GTP−RafRBD. The binding affinity of RafRBD to H-RasWT−GDP is ∼1000fold reduced compared with that of RafRBD to H-RasWT− GTP.313 Filchtinski et al.314 previously used a computational protein design approach to redesign the binding interface on RafRBD to optimize its interactions with the inactive H-Ras− GDP state. On the basis of the binding interface between Rap1AE30D/K31E−GppNHp and RafRBD, six mutations, including F61W, R67L, V69E, N71R, K84R, and V88I, on the RafRBD with the binding interface were predicted to enhance the intermolecular interactions between mutant RafRBD and H-Ras−GDP. The calculated binding affinity showed that four mutations, R67L, N71R, K84R, and V88I, would improve the binding potency of mutant RafRBD to H-Ras−GDP and that all mutations on RafRBD, with the exception of V69E, would increase the potency of H-Ras−GDP against H-Ras−GppNHp, which suggest that these mutations, except V69E, shift the binding specificity to H-Ras−GDP. The guanine nucleotide dissociation inhibitor (GDI) assay revealed that the designed RafRBD mutant containing all six mutations was the largest shift of specificity in favor of H-Ras−GDP against H-Ras− GppNHp by ∼10-fold and the single F61W and N71R mutant, the double F61W/N71R mutant, the triple F61W/N71R/W88I

mutant, and the quadruple F61W/R67L/N71R/W88I mutant shifted the specificity in favor of H-Ras−GDP by ∼2−3-fold. A85K RafRBD is a previously reported mutation that markedly shifts the binding specificity toward H-Ras−GDP (4.2-fold). Through the combination of the double N71R/A85K mutations of RafRBD, the GDI assay exhibited that this double mutant led to a ∼12-fold shift of binding specificity toward HRas−GDP, with the shifted potency similar to that of the RafRBD mutant with all six F61W/R67L/V69/N71R/K84R/ W88I mutations. The crystal structures of RafRBDA85K bound to H-RasWT−GDP and of RafRBDN71R/A85K bound to HRasWT−GDP were solved. Backbone superimposition of the structure of H-RasWT−GDP−RafRBDA85K (PDB ID 3KUD) onto those of H-RasWT−GppNHp−RafRBD (PDB ID 4G0N) and H-RasWT−GDP (PDB ID 4Q21) (Figure 41A) and of HRasWT−GDP−RafRBDN71R/A85K (PDB ID 3KUC) onto those of H-RasWT−GppNHp−RafRBD and H-RasWT−GDP (Figure 41B) reveals that the conformation of the switch I region of HRasWT−GDP in both mutant H-Ras−RafRBD complexes is very similar to that in H-RasWT−GppNHp−RafRBD rather than to that in the unbound H-RasWT−GDP. Further comparison of the relative B-factors of the switch I region in the structures of H-RasWT−GDP−RafRBDA85K, H-RasWT− GDP−RafRBDN71R/A85K, Rap1AE30D/K31E−GppNHp−RafRBD, and H-RasWT−GppNHp−RafRBD reveals that the flexibility of the switch I region of H-Ras is markedly increased in the GDPbound state compared to the GppNHp-bound state, suggesting that the flexibility of the switch I region of Ras plays a critical role in determining the Raf specificity for Ras−GTP or Ras− GDP. An analysis of the binding interface shows that the interface between H-RasWT−GDP and mutant RafRBD is different from that between H-Ras WT −GppNHp and RafRBDWT. For example, in the interface of H-RasWT−GDP− RafRBDA85K complex (Figure 41C), the strong salt bridges are from E31Ras−K84Raf and D38Ras−R89Raf and the weak salt bridges are from D33Ras−K85Raf and D38Ras−K85Raf. In the interface of H-RasWT−GDP−RafRBDN71R/A85K complex (Figure 41D), the strong salt bridges are from E31Ras−K84Raf, D33Ras− R71Raf, E37Ras−R59Raf, and D38Ras−R89Raf and the weak salt bridges are from D33Ras−K85Raf and E37Ras−R67Raf. The total salt bridges in the binding interface of H-RasWT−GDP− RafRBDN71R/A85K complex outweigh those of H-RasWT−GDP− RafRBD A 8 5 K . As expected, the binding affinity of RafRBDN71R/A85K to H-RasWT−GDP (0.442 μM) is more potent than that of RafRBDA85K to H-RasWT−GDP (1.7 μM). 6640


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RafCRD complex have not been successful, rendering the structural basis of how RafCRD interacts with Ras unclear. 11.2. Interaction with PI3K

The lipid kinase family of PI3Ks plays a pivotal role in many cellular processes, encompassing proliferation, cell survival, differentiation, and metabolism.315−317 Class I PI3Ks, the best physiologically, biochemically, and structurally characterized kinase of PI3K family, consists of four isoforms, α, β, γ, and δ.318 Each isoform is a heterodimer that comprises a p110 catalytic subunit and a p85 regulatory subunit.319,320 The overall organization of the p110 catalytic subunit is highly conserved (Figure 42A), including an N-terminal adaptorbinding domain (ABD) (residues 34−141), a Ras binding domain (RBD) (residues 217−309), a C2 domain (residues 357−521), a helical domain (residues 541−723), and a catalytic domain (residues 828−1073). Biochemical studies have welldocumented that all class I PI3Ks bind Ras in a GTPdependent manner.321−324 However, the binding affinity of PI3Ks to Ras is significantly weaker than that of Raf to Ras. For example, the dissociation constant Kd for PI3KγWT to HRasWT−GppNHp is 3.2 ± 0.5 μM,163 which is higher than the 0.018 μM K d for the H-Ras WT −GppNHp−RafRBD WT complex.194 In 2000, Pacold et al.163 solved the first cocrystal structure of the complex between the oncogenic H-RasG12V bound to GppNHp and the catalytic subunit of PI3KγV233K (PDB ID 1HE8). The V233K mutation of PI3Kγ was introduced in order to increase the binding affinity of mutant PI3Kγ to H-Ras, thereby facilitating cocrystallization with H-Ras. In the structure of H-RasG12V−GppNHp−PI3KγV233K complex, the electron density for the ABD of PI3KγV233K is not visible. H-RasG12V− GppNHp interacts with the RBD of PI3KγV233K (Figure 42B). In the binding interface between H-Ras and PI3Kγ, PI3Kγ interacts mainly with the switch I region of H-RasG12V, and the

Figure 41. (A) Cartoon representation of the backbone superimposition of the structure of H-RasWT−GDP−RafRBDA85K (PDB ID 3KUD, pink) onto that of H-RasWT−GppNHp−RafRBD (PDB ID 4G0N, blue) and H-RasWT−GDP (PDB ID 4Q21, cyan). (B) Backbone superimposition of the structure of H-RasWT−GDPRafRBDN71R/A85K (PDB ID 3KUC, pink) onto that of H-RasWT− GppNHp−RafRBD (blue) and H-RasWT−GDP (cyan). (C) The binding interface between H-RasWT−GppNHp and RafRBDA85K. Salt bridges are represented by blue dotted lines. (D) The binding interface between H-RasWT−GppNHp and RafRBDN71R/A85K.

The RafCRD interaction with Ras−GTP is required for Raf activation. However, to date, attempts to crystallize the Ras−

Figure 42. (A) Scheme of the domain organization of PI3K p110γ catalytic domain. (B) Surface representation of the H-RasG12V−GppNHp− PI3KγV233K complex (PDB ID 1HE8). The H-Ras is colored orange and GTP is depicted by stick models. The PI3Kγ RBD domain is colored pink, the PI3Kγ C2 domain is colored cyan, the PI3Kγ helical domain is colored yellow, the PI3Kγ kinase domain is colored blue, and the linker is colored gray. (C) The salt bridges or hydrogen-bonding interactions in the switch I·PI3Kγ−RBD interface. Salt bridges or hydrogen bonds are represented by blue dotted lines. (D) Cartoon representation of the H-RasG12V−GppNHp−PI3KγV233K complex. The switch I and switch II regions of H-Ras are colored red and green, respectively. (E) The salt bridge and hydrophobic interactions in the switch II·PI3Kγ−RBD interface. (F) Backbone superimposition of the structure of H-RasG12V−GppNHp−PI3KγV233K (pink) onto that of free PI3Kγ−ATP (PDB ID 1E8X, cyan). H-RasG12V is colored orange. GppNHp and ATP are depicted by stick models. 6641


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strand β2 of H-RasG12V forms an antiparallel β-sheet with the β2 of PI3KγV233K. These structural features are also shared by the H-Ras/Rap1A−RafRBD complexes.162,310 The interaction of the switch I region of H-RasG12V with the PI3Kγ−RBD contributes to the ordering of a loop, residues 255−267, in the RBD. The electron density for this loop is invisible in the uncomplexed PI3Kγ structure. The hydrogen-bonding or salt bridges interactions between the switch I region of H-RasG12V and PI3Kγ include D33Ras−K251PI3Kγ, D33Ras−K255PI3Kγ, E37Ras−K223PI3Kγ, E37Ras−T232PI3Kγ, and D38Ras−Q231PI3Kγ (Figure 42C). Site-directed mutagenesis confirmed that all T232D, K251A/E and K255A mutations on PI3Kγ abolish binding to H-Ras. These pieces of evidence can be explained by all mutations disrupting hydrogen donors or acceptors and the T232D and K251E mutations further introducing charge repulsion in the Ras switch I·PI3Kγ interface. Unexpectedly, the unique feature of the switch II region of H-RasG12V interacting with the PI3Kγ is observed in the H-RasG12V− GppNHp−PI3KγV233K complex (Figure 42D), but it is not seen in the H-Ras/Rap1A−RafRBD complexes. Indeed, the engagement of the switch II region of H-Ras with its upstream regulators has previously only been observed in the crystal structures of H-Ras−GAP and H-Ras−SOS complexes.193,272 In the Ras switch II·PI3Kγ interface (Figure 42E), the ionic interactions include E63Ras−K234PI3Kγ and Y64Ras−K234PI3Kγ. Moreover, Y64 on the switch II region of H-Ras forms hydrophobic interactions with residue F221 of PI3Kγ. Sitedirected mutagenesis validated that both F221S and K234A mutations of PI3Kγ decrease the binding affinity of mutant PI3Kγ to H-Ras, supporting the switch II region of H-Ras engaging in the interaction with PI3Kγ. Further backbone superimposition of the structure of H-RasG12V−GppNHp− PI3KγV233K onto that of free PI3Kγ−ATP (PDB ID 1E8X)325 shows that compared with the free PI3Kγ, the conformational changes of PI3KγV233K in response to H-Ras binding take place mainly in the C2 domain and the C-terminal lobe of the kinase domain (Figure 42F), which are distal to the H-Ras·PI3Kγ interface. The conformational changes of PI3Kγ upon Ras binding suggest that an allosteric mechanism is involved in the activation of PI3K. Recent work revealed that the direct interaction of Ras with the PI3K p110α catalytic subunit is required for tumor-induced angiogenesis.326−329 To date, the cocrystal structures of the complex between Ras and p110α are still unavailable. As a result, the structural basis of how Ras interacts with p110α remains unresolved. 11.2.1. Calmodulin’s Full Activation of PI3Kα in the Case of Oncogenic K-Ras4B. Calmodulin was shown to directly activate PI3Kα and it is proposed to form a complex with K-Ras4B and PI3Kα.330,331 Normally, PI3Kα is recruited to the membrane with the help of activated tyrosine kinase receptors (RTKs). However, as we suggested recently, when KRas4B is activated by oncogenic mutations, calmodulin can substitute for the RTK signal, to achieve full activation of the PI3Kα.332,333 It is a uniquely highly positively charged HVR and the fact that it is only farnesylated makes K-Ras4B the only Ras isoform able to bind calmodulin, although recently we proposed that the depalmitoylated form of K-Ras4A, the HVR of which is also positively charged, albeit not to the same extent as that of K-Ras4B, may also bind.93 The presence of an additional post-translational modification such as palmitoyl could sterically impede calmodulin’s binding. The main driving force for the calmodulin−K-Ras interaction is the electrostatic

interaction between calmodulin and the HVR; however, the docking of farnesyl into a calmodulin pocket further stabilizes the interaction.198,334 The binding of calmodulin to the HVR of GTP-bound K-Ras4B shifts the equilibrium from membranebound K-Ras4B to the unbound state. Due to its activation of the PI3Kα/Akt pathway in the absence of a signal from RTK, calmodulin plays a critical role in K-Ras-driven cancers that is not the case in cancers driven by other Ras isoforms.332,333 The high calcium levels observed in these cancers may explain calmodulin’s role in recruiting and activating PI3Kα. Without RTK signals, oncogenic mutations in K-Ras can activate PI3Kα, but they are unable to achieve full activation; thus, they may lead to oncogene-induced senescence (OIS) or to proliferation and differentiation.335 Calmodulin can fully activate PI3Kα through two mechanisms: first, binding to the nSH2 domain of PI3Kα substituting for the pYXXM motif of activated RTKs this action relieves the steric autoinhibition action of the nSH2 domain on the catalytic p110 subunitand, second, acting to allosterically activate PI3Kα through binding to the cSH2 domain of its p85 subunit. Using PRISM336−338 we modeled a trimer, K-Ras4B−GTP/CaM/PI3Kα (Figure 43), with an interaction between calmodulin and cSH2-p85α. A second calmodulin molecule can bind the nSH2 domain. Calmodulin is abundant in these cancer tissues. In addition to its activation of PI3Kα, calmodulin also temporally downregulates Raf’s activation, likely through its action of shifting the K-Ras4B equilibrium toward its membrane-unbound state.332,333 Calmodulin’s actions may help clarify the more profound consequences of mutant K-Ras4B in human cancers, particularly pancreatic, lung, and colorectal adenocarcinomas, as compared to other Ras isoforms. 11.3. Interaction with RalGDS

To date, four different members of the RalGDS family have been identified as binding partners for small G-proteins: RalGDS, RGL (RalGDS like), RGL2 (RalGDS like-2)/Rlf (RalGDS like factor), and RGL3 (RalGDS like-3).339 RalGDS is one of several known GEFs that function by activating Ral A and B GTPases.340 Ral interacts with its effectors, such as Sec5, Filamin, RalBP1, and ZONAB, in the regulation of endocytosis, exocytosis, and actin organization and in the control of gene expression.341 In 1994, on the basis of a yeast two-hybrid system, several independent groups found that RalGDS is a putative Ras effector that interacts with the active GTP-bound Ras.342−344 Moreover, the authors revealed that the RalGDSRBD and RafRBD compete for binding to Ras or Ras-like GTPases, suggesting that RalGDS-RBD and RafRBD may bind to a similar Ras interface. On the basis of these results, Miller et al.345 further pinpointed that RalGDS functions as an effector of Ras in cAMP-mediated growth stimulation. In 2005, GonzálezGarciá et al.346 showed that mice lacking RalGDS presented reduced tumor incidence, size, and progression in a skin cancer model that is induced by the oncogenic H-Ras mutant. This significant finding suggests that RalGDS plays an essential role in Ras-dependent carcinogenesis in vivo. However, the relation of RalGDS activation to human cancer remains to be validated and better understood.347 All members of the RalGDS family contain three domains, encompassing an upstream Rem, a central Cdc25 homology domain, and a C-terminal RBD. RalGDS proteins serve as an exchange factor by virtue of the Cdc25 homology domain, similar to the RasGEF SOS. The RalGDS-RBD involves the GTP-dependent interaction with Ras and Ras-related proteins. 6642


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that the orientation of RalGDS-RBD with respect to H-Ras is clearly different from that of RafRBD to H-Ras, leading to the 35° tilt in the binding angle of RalGDS-RBD compared to RafRBD when bound to H-Ras (Figure 44C). Recently, using vibrational Stark effect spectroscopy and MD simulations, Walker et al.348 unearthed the conserved electrostatic field at the Ras−effector interface as a driving force that results in two structurally identical effector proteins Raf and RalGDS adopting a distinct orientation in the lowest-energy interaction with Ras. The salt bridges or hydrogen-bonding interactions in the switch I·RalGDS-RBD interface include K31Ras−D51RalGDS, K31Ras−N54 RalGDS, K31 Ras−D56 RalGDS, D33 Ras−K52 RalGDS, P34 Ras −K52 RalGDS , T35 Ras −K52 RalGDS , E37 Ras −R20 RalGDS , E37Ras−Y31RalGDS, and D38Ras−S33RalGDS (Figure 44D). A previous mutational study of H-Ras at the switch I−effector interface showed that the E37G mutation of H-Ras enables binding to RalGDS but not to Raf or PI3K and that T35S mutation of H-Ras abolishes the binding to RalGDS but does not eliminate the interaction with RafRBD.349−353 These differences are ascribed to the distinct interaction between HRas and RalGDS compared to that between H-Ras and RafRBD. For example, E37 of H-Ras does not interact with RalGDS-RBD, but it forms salt bridges with residues R59 and R67 of RafRBD. T35 of H-Ras interacts with the residue K52 of RalGDS-RBD through a water-mediated hydrogen bond, but it does not interact with RafRBD. These differences may play key roles in effector selectivity when Ras is constrained by a preferred interaction state with respect to the membrane. At the switch II·RalGDS-RBD interface, the intermolecular interaction is formed between the helix α2 of the switch II region of H-Ras and strand β2 and helix α2 of another RalGDSRBD (Figure 44B). The association of RalGDS-RBD with the switch II region of H-Ras is not observed in the Ras−RafRBD complex. However, this structural feature may be shared by the Ras−RafCRD complex, because RafCRD also binds to the switch II region of Ras. The salt bridges or hydrogen-bonding interactions in the switch II·RalGDS-RBD interface include E63Ras−L35RalGDS, E63Ras−K48RalGDS, and Q70Ras−Q39RalGDS (Figure 44E). Mutagenesis showed that a G60A mutation of H-Ras abrogates the binding of mutant H-Ras to RalGDS-RBD as a consequence of the impact of this mutation on the conformation of the switch II region of H-Ras.354

Figure 43. A K-Ras4B−GTP/calmodulin (CaM)/PI3Kα ternary complex model based on the prediction. We used the G-domain of K-Ras (166 residues), full length CaM (149 residues), and the p110 catalytic p85 regulatory subunits of PI3K as target proteins. Full-length CaM has about 75 structures in the PDB. We considered only X-ray structures with

Ras Conformational Ensembles, Allostery, and ... - ACS Publications

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