Functional Role and Hierarchy of the Intermolecular Interactions in

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Functional Role and Hierarchy of the Intermolecular Interactions in Binding of Protein Kinase Clients to the Hsp90-Cdc37 Chaperone: Structure-Based Network Modeling of Allosteric Regulation Gabrielle Stetz, and Gennady M. Verkhivker J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.7b00638 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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Journal of Chemical Information and Modeling

Functional Role and Hierarchy of the Intermolecular Interactions in Binding of Protein Kinase Clients to the

Hsp90-Cdc37

Chaperone:

Structure-Based

Network Modeling of Allosteric Regulation Gabrielle Stetz1, Gennady M. Verkhivker1, 2 ‡ 1

Graduate Program in Computational and Data Sciences, Department of Computational Sciences,

Schmid College of Science and Technology, Chapman University, One University Drive, Orange, CA 92866, USA 2

Chapman University School of Pharmacy, Irvine, CA 92618, USA

‡corresponding author E-mail: [email protected]

1 ACS Paragon Plus Environment

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Abstract A fundamental role of the Hsp90-Cdc37 chaperone machinery in mediating conformational development and activation of diverse protein kinase clients is essential for signal transduction. Structural and biochemical studies have demonstrated that characterization of global conformational changes and allosteric interactions in the Hsp90-Cdc37-kinase complexes are central to our understanding of the mechanisms underlying kinase recruitment and processing by the Hsp90-Cdc37 chaperone. The recent cryo-electron microscopy structure of the Hsp90Cdc37-Cdk4 kinase complex has provided a framework for dissecting regulatory principles underlying differentiation and recruitment of protein kinase clients to the chaperone machinery. In this work, we have characterized functional role and hierarchy of the intermolecular interactions in binding of protein kinase clients to the Hsp90-Cdc37 system. The network analysis revealed important relationships between structural stability, global centrality and functional significance of regulatory hotspots in chaperone regulation and client recognition. A unique asymmetric topography of the intermolecular communities in the chaperone-kinase complex has quantified a central mediating role of Cdc37 in client recognition and allosteric regulation of the chaperone-kinase complex. Modeling of allosteric pathways in the chaperone complex has further clarified structural and energetic signatures of allosteric hotspots, particularly linking sites of post-translational modifications in Hsp90 with their role in allosteric interactions and client regulation. The results of this integrative computational study are compared with a wide range of structural, biochemical and cell-based experiments, offering a robust network-centric model of allosteric regulation and client kinase recognition by the Hsp90Cdc37 chaperone machine.


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Introduction The 90 kDa heat-shock proteins Hsp90s are evolutionary conserved ubiquitous molecular chaperones that are vital for functioning of all eukaryotic organisms.1,2 The Hsp90 chaperones assist in folding of protein substrates, maintaining stability and activity of numerous signaling client proteins associated with cell growth, proliferation, differentiation, and cell death.3-7 The Hsp90 family consists of four different isoforms: cytosolic constitutive (β) and inducible (α) forms, a mitochondrial member (TRAP1) and an ER-resident protein (GRP94). The monomers in the Hsp90 homodimer feature functional domain organization.

The N-terminal domain

(Hsp90-NTD) is involved in the nucleotide binding and ATP hydrolysis, the middle domain (Hsp90-MD) is often implicated in binding of cochaperones and recognition of client proteins, and the

is involved in constitutive dimerization.8-16

C-terminal domain (Hsp90-CTD)

Crystallographic and biophysical studies have characterized conformational landscape and structural transformations of Hsp90 that are associated with the ATP binding and hydrolysis cycle.17-21 Hydrogen/deuterium exchange mass spectrometry (HX-MS) experiments have revealed conformational plasticity of eukaryotic Hsp90s that are considerably more dynamic than the HtpG chaperones. Importantly, these studies have demonstrated that conformational dynamics of the eukaryotic cytosolic Hsp90 is largely independent on the nucleotide binding and can be mainly driven by stochastic thermal fluctuations.22-24 The cytosolic Hsp90 proteins utilize assistance of cochaperones in managing protein clients in the course of the ATPase cycle.25-27 The Hsp90 interactions with client proteins can be also regulated through a multitude of posttranslational modification (PTM) sites that are broadly distributed across the Hsp90 domains.28-30 Cdc37 is a client-specific cochaperone that assists Hsp90 in managing conformational folding, maturation and activation of protein kinases.,31 3 ACS Paragon Plus Environment


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The fundamental mechanisms and thermodynamics of the Hsp90-client interactions were first explored in a seminal cell-based interactome mapping study32 revealing that the Hsp90-Cdc37 machinery can recognize kinase clients by sensing their conformational instability, while rejecting stable native folds presented to the chaperone by nonclient kinases. HX-MS studies and functional assays of structurally similar kinase chimeras have revealed that the Hsp90-Cdc37 system can differentiate between weak c-Src kinase client and strong constitutive client v-Src kinase.33,34 According to these studies, the interaction strength of chaperone-client interactions correlates with the degree of unfolding cooperativity and solvent exposure of the regulatory regions in the kinase catalytic domain.33,34 The emerging mechanism of the Hsp90-Cdc37 client recognition implies that Cdc37 may function as a principal gate-keeper to the Hsp90 system by recognizing conformational instability of client kinases. Cdc37 plays a central role in chaperoning of kinase clients

since

impairment of Cdc37 function can dramatically affect

binding of oncogenic kinase clients with the Hsp90-Cdc37 chaperone system.35 NMR spectroscopy studies have shown that Cdc37 facilitates binding of client substrates by eliciting a cascade of

concerted conformational changes and orchestrating

an allosteric cross-talk

between multiple interaction sites of the interacting proteins.36,37 However, atomistic details and mechanics of client processing by the Hsp90-Cdc37 chaperone system have been poorly understood until very recently. The preliminary EM reconstruction of the Hsp90-Cdc37-Cdk4 kinase complex indicated an asymmetric intertwined topology of multiprotein assembly, but the atomistic details of the binding interfaces hypothetical,

and functional interactions remained largely

offering different and often conflicting explanations of the underlying

mechanism.38 The latest cryo–electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex has produced shock waves in the chaperone community, 4 ACS Paragon Plus Environment

unveiling a completely

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unexpected structural arrangement, in which the Hsp90 and Cdc37 trap a partially disordered kinase state, providing framework for understanding allosteric mechanisms of kinase recruitment to the chaperone.39,40

Allosteric regulation of the Hsp90 interactions

with

cochaperones and

binding partners

determines versatility of the chaperone system in coordinating signaling responses and timely processing of

multiple clients. Computational studies of allosteric signaling in single domain

proteins and complex multiprotein assemblies have gained a significant momentum in recent years41-49 reshaping the conventional view of allostery. The statistical thermodynamics-based model of allosteric regulation has clarified many aspects of population-shift and dynamic-based mechanisms of allostery50-52 suggesting that protein ensembles of preexisting conformational states and communication pathways can be modulated through allosteric interactions with substrates and binding partners.53 Recent investigations from our laboratory and several other groups have suggested that Hsp90 interactions with cochaperones and client proteins can be highly dynamic and cooperative in nature, leading to modulation of conformational stability in the clients and eliciting global conformational rearrangements in the chaperones.54-62 These studies indicated that mediating sites in the Hsp90 interaction networks may signify hotspots of chaperone regulation.

Despite significant insights in biochemical and structural characterizations of the Hsp90-Cdc37 interactions with the diverse kinase clientele, the mechanism by which allosteric cross-talk of the binding interfaces promotes integration of client kinases to the Hsp90 system remains elusive. The recent reevaluation of the role of the Hsp90 chaperones in the kinase lifecycle has pointed to 5 ACS Paragon Plus Environment


several conceptual issues concerning regulation of the Hsp90-Cdc37 interactions with client proteins.40 Some of these outstanding questions include a) detailing of functional specialization of Cdc37 N-terminal (Cdc37-NTD) and C-terminal domains (Cdc37-M/C) in recruitment of partially unfolded kinase clients; b) quantifying allosteric cross-talk and hierarchy of the intermolecular binding interfaces in the Hsp90-Cdc37-client complexes; c) clarifying regulatory role of functional sites in allosteric mechanisms of Hsp90-Cdc37 binding with kinase clients; c) characterizing allosteric communication pathways and mediating centers of allosteric signaling in the Hsp90-client complexes. In the current study, we combined molecular simulations and protein stability analysis with modeling of the interaction networks and communication pathways to characterize

allosteric

interactions in the Hsp90-Cdc37-kinase complex and

identify regulatory hotspots that control client recognition by the chaperone machine. The incorporation of both dynamic residue correlations and coevolutionary residue dependencies provided a novel theoretical framework for

reconstruction of allosteric interaction networks

and communication pathways in the Hsp90 structures. A community analysis reveals modularity of allosteric interaction networks and hierarchy of binding interfaces. By identifying principal stabilizing communities and communication pathways at the atomic details, we obtain novel molecular insights into regulatory role and coupling of conserved PTM sites that serve as allosteric switch points of the Hsp90-Cdc37 system. The results of this investigation provide a novel rationale to the latest experimental data on client recruitment by the Hsp90-Cdc37 system, detailing how Hsp90 and Cdc37 act cooperatively in protecting unstable kinase clients and restraining their undesirable oncogenic activity.


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Materials and Methods Discrete Molecular Dynamics and All-Atom Reconstruction of Conformational Ensembles

The structures of the human Hsp90-Cdc37-Cdk4 complex (pdb id 5FWM, 5FWK, 5FWL) were obtained from the Protein Data Bank (www.rcsb.org). During structure preparation process, hydrogen atoms and missing residues were initially added and assigned according to the WHATIF program web interface.63 The unresolved segments in the Hsp90-Cdc37-Cdk4 structures were modeled and reconstructed using template-based loop prediction approach ArchPRED.64 We employed the formalism and implementation of the discrete molecular dynamics (DMD) simulations65,66 to rapidly generate conformational ensembles based on structures of the human Hsp90-Cdc37-Cdk4 complex. In the DMD approach, the protein structures were modeled as systems consisting of C α residue-based beads interacting through a discontinuous square well potential. The details of the DMD model implemented in this study were outlined in our earlier investigations.67

Conformational ensembles generated in DMD

simulations were subsequently subjected to all-atom reconstruction by PULCHRA method68 and CG2AA tool.69 The all-atom conformations were optimized using 3Drefine method70 that utilizes atomic-level minimization with a composite physics and knowledge-based force fields. Protein Stability Calculations A systematic alanine scanning of protein residues was performed using FoldX approach.71 We utilized a graphical user interface for the FoldX calculations72 that was implemented as a plugin for the YASARA molecular graphics suite package.73 To obtain ensemble-based estimates of protein stability changes and ensure efficiency of FoldX computations, we evaluated the



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average ∆∆G values using representative samples (~200-300) of the equilibrium ensembles within a modified FoldX protocol.74 Protein Structure Networks and Modeling of Communication Pathways For network-based analysis, we employed a graph-based representation of protein structures in which residues as network nodes and inter-residue edges represent residue interactions.75-77 The details of graph construction using residue interaction cut-off strength ( Imin ) were presented in our previous studies of molecular chaperones.78-80. The edges in the residue interaction network are weighted based on coevolutionary mutual information80 and dynamic residue correlations couplings obtained from molecular simulations.81 in this model, weight wij is defined as the element of a matrix measuring the generalized correlation coefficient rMI ( x i , x j ) between residue fluctuations in structural and coevolutionary dimensions. The composite residue vector describes structural residue positions and respective proximity-based coevolutionary score80: wij = − log[rMI ( x i , x j )] (1)

Mutual Information (MI) analysis is used to estimate the extent of the mutual coevolutionary relationship between pairs of positions in the protein family.82,

83

MI was calculated using

MISTIC approach.83 The edge lengths in the residue interaction network are obtained using the generalized correlation coefficients rMI ( x i , x j ) associated with both dynamic correlation extracted from DMD trajectories and mutual information shared by each pair of residues. The length (i.e. weight) of the edge that connects nodes i and j wij = − log[rMI ( x i , x j )] is calculated from the generalized correlation coefficient between this nodes.80 The ensemble of shortest paths is determined from matrix of communication distances by the Floyd-Warshall algorithm84 that compares all possible paths between each pair of residue nodes. 8 ACS Paragon Plus Environment

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Network Centrality Analysis and Community Detection Using the constructed protein structure networks, we computed the residue-based betweenness parameter. The betweenness of residue i is defined to be the sum of the fraction of shortest paths between all pairs of residues that pass through residue i :

N

Cb (ni ) = ∑ j 2.0 kcal/moll in these positions signified a central functional role of this interfacial hotspot. Protein stability changes in the Cdk4 kinase revealed differential stabilization of the kinase lobes, which is manifested in a structurally stable C-lobe and a more flexible N-lobe. Indeed, the N-lobe residues showed a greater tolerance to alanine substitutions, while mutations of the C-lobe residues produced a more significant destabilization effect (Figure 3D). In the complex, energetic polarization of the kinase lobes was even more apparent, revealing only small changes in protein stability caused by mutations of the N-lobe residues. This effect was particularly pronounced in the partially disordered N-lobe regions: the αC-helix (residues 50-67), αC-β4 loop (residues 67-74) and a β4-β5 sheet (residues 75-95). In contrast, substitutions in the C-lobe regions resulted in the markedly larger energetic changes, which were comparable with the corresponding effects in the folded kinase form (Figure 3D). Hence, client recruitment into the Hsp90-Cdc37 chaperone may enhance energetic instability of the N-lobe, while maintaining rigidity of the C-lobe. The observed differential stabilization of the kinase lobes may reflect the intrinsic dynamic preferences of kinase clients that can be further amplified 17 ACS Paragon Plus Environment


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upon integration into the chaperone system. These findings are consistent with several recent studies suggesting that the excessive conformational

mobility of the N-lobe regions

in

oncogenic kinases can shape up structural preferences of activation cancer mutations often populating these regions which allows for robust modulation of functional dynamics.99, 100 Structural mapping highlighted the fact that high stability sites in the Hsp90 dimer were often spatially proximal and formed tight interaction modules. Some of these hydrophobic clusters stability hotspots (I288-F304-L332 and P287-W289-F304-Y356) were immediately adjacent to the NTD-MD regions (L388, I390 and R392 residues), facilitating the inter-domain interactions and rigidifying the catalytic site environment (Figure 4A, B). Structural analysis of stability hotspots also illustrated the interconnectivity of these centers in the complex (Figure 4). Several highly stable residues are assembled into an interacting cluster (I404-V381-I408-L369-I370L401) that is directly linked to the interfacial lysine residues (K399, K402, K406, K411) making specific interactions with Cdc37-NTD (Figure 4 C). Another hotspot of protein stability is formed by the residue cluster F376-L343-F433-N436 that anchored the Src-loop at the binding interface with the kinase N-lobe (Figure 4D). These clusters include or directly connected with several regulatory centers. In particular, a significant number of conserved PTM sites (K402, K406, K411, K438) are strategically positioned to couple the energetic hotspot centers in Hsp90MD with Cdc37-NTD and Cdk4 N-lobe regions (Figure 4C,D). We argue that structural preferences of these PTM centers in Hsp90 may be related with their fundamental role as dynamic carriers of allosteric signaling and mediators of binding interactions in the chaperoneclient complexes. In general, we found that stable energetic centers in the Hsp90 chaperone are coupled with more flexible interfacial residues that become immobilized in the chaperone-client complex. Our findings corroborated with the evidence that regions of high and low structural 18 ACS Paragon Plus Environment

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stabilities could be often proximal that enables coupling between global conformational changes and binding to protein substrates. The observed partitioning of stable and flexible regions in the chaperone-kinase complex is consistent with the idea that protein ability to evolve new functions may be linked with structural modularity, whereby flexible recognition regions are linked to a highly ordered central scaffold.101,102 This pattern was observed in the Hsp90-Cdc37-Cdk4 complex, where the central Hsp90-MD core is enriched by highly stable energetic hotspots, while flanking recognition loops are flexible in the unbound form and become ordered in the chaperone-kinase complex.



Figure 4. Structural mapping of protein stability hotspot clusters. (A) The overview of the intermolecular interfaces and energetic hotspot centers in the Hsp90-Cdc37-Cdk4 complex. Hsp90-A monomer is shown in orange ribbons and Hsp90-B monomer B is in pink ribbons. Cdc37 is in red ribbons, and Cdk4 kinase is in yellow ribbons. The location of three major centers of energetic hotspots is indicated by rectangular boxes colored in red (with a close-up in panel B), blue (a close-up is presented in panel C), and green (a close-up is in panel D). (B) Structural mapping of the intramolecular cluster connecting energetic hotspots near the NTD-MD interface. For clarity only Hsp90-B monomer (colored in pink ribbons) is shown. The high stability clusters I288-F304-L332 and P287-W289-F304-Y356 are shown in domaincolored spheres. These residues are directly linked to L388, I390 and R392 (in sticks) that bridge NTD and MD regions near the catalytic site. Catalytic residue R392 and ATP are shown in atomcolored spheres. (C) A close-up of the protein stability hotspot formed by the hydrophobic cluster (I404-V381-I408-L369-I370-L401). The hotspot residues are shown in domain-colored pink spheres (Hsp90-B monomer of the dimer). This stability hotspot is immediately proximal to the Hsp90 binding residues K399, K402, R405, K406 (shown in pink sticks ) that form a network of salt bridges with Cdc37-NTD residues D14,D15, E16,D17,E18 ( in red sticks). Hsp90-B in pink ribbons, Cdc37 in red ribbons, and Cdk4 is in yellow ribbons. (D) The protein stability hotspot cluster F376-L343-F433-N436 (in pink spheres) anchors the Src-loop residues R338, F341, L343, F344 (in sticks) that make contacts with the Cdk4 N-lobe regions. The Cdc37-M/C domain is shown in red ribbons and Cdk4-N lobe is shown in yellow ribbons.


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Network Analysis Identifies Mediating Centers of Allosteric Interactions To investigate allosteric coupling of the binding interfaces and identify global mediating centers that control kinase client recruitment, we characterized the residue interaction network in the Hsp90-Cdc37-Cdk4 complex. Using a graph-based representation of protein structures77, we integrated topological connectivity of protein residues and dynamic contact maps of residue cross-correlations with coevolutionary residue dependencies in the construction of the residue interaction network.80 A global centrality parameter, residue betweenness (also termed residue centrality in the text), was explored to identify mediating centers of allosteric interactions in the unbound and bound Hsp90 structures. The majority of high centrality residues were located in the Hsp90-MD regions and assembled in tight interaction clusters, likely to strengthen robustness of key mediating centers (Figure 5A, B). The notable peaks in the Hsp90-MD corresponded to W289, F341, L343, Y356, I370, Y373, F376, I400, F433, E515, and D518 residues. These sites coincided with major stabilization centers, suggesting that a small group of hotspot clusters may be involved in mediating global interaction network. To determine client-induced modulation of the global interaction network, we evaluated differences in the residue centrality of the Hsp90 residues in the unbound and client-bound forms of Hsp90 (Figure 5 C, D). Several peaks in the Hsp90-MD regions were induced upon complex formation, particularly a pronounced increase in the network centrality of the Src loop (residues 341-350) involved in the interface with the disordered kinase regions (Figure 5D). These high centrality sites also overlapped with the important stabilization clusters in this region (F341-L343-F344-F376 and F376-L343-F433N436). Another noticeable change in the network organization of the complex was a markedly increased centrality of the CTD helix 15 (residues 599-611).



Figure 5. Residue centrality profiles of the Hsp90 dimer. Residue-based centrality distributions of the Hsp90 monomers in the chaperone-kinase complex (A, B). The client-induced differential changes in the residue centrality of the Hsp90 residues (C, D) are obtained from differences between residue betweenness values in the complex and unbound Hsp90 dimer. The distributions are derived by averaging computations of network parameters over equilibrium ensembles extracted from DMD trajectories. The distributions are shown in domain-colored bars. The Hsp90-NTD residues are shown in red bars, Hsp90-MD residues are in blue bars, and Hsp90CTD residues are in green bars.


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Hence, kinase recruitment may induce significant changes in the network centrality of the Hsp90 recognition regions that could serve as mediators of allosteric interactions. The residue centrality profiling of the Cdc37 and Cdk4 domains pointed to the key mediating role of the Cdc37-NTD in the allosteric interaction network. Two pronounced sharp peaks were seen in the Cdc37-NTD profile (Figure 6A). The first peak corresponded to cluster of residues (D14, D15, E16, D17, and E18) involved in the interface with Hsp90-B monomer. The second peak (residues H20, P21, N22, I23, F29, W31, and N32) corresponded to the binding interface formed by Cdc37-NTD with the Cdk4 C-lobe (residues 147-153). The importance of this interface was also reflected in the centrality distribution of Cdk4 client, featuring peak near C-lobe residues 147-153 (Figure 6B). These profiles reflected the importance of the Cdc37-NTD/Cdk4 N-lobe interface in mediating allosteric communications.

The observed rewiring of the global interaction network near the Hsp90 intermolecular interfaces may be linked with dynamic exchanges between the Hsp90 and Cdk4 that manifested in stabilization of the Hsp90-CTD binding regions involved in protection of unstable kinase client. Our observations also suggested that the hierarchy of spatially separated binding interfaces may be important for allosteric mechanism of client recruitment. According to the network analysis, Cdc37-NTD/Cdk4 C-lobe and Hsp90-Cdk4 N-lobe interfacial regions are central for client binding, whereas binding interface formed between Cdc37-M/C and Cdk4 N-lobe regions may play a supporting role. In this mechanism, Cdc37-NTD and Hsp90 can act cooperatively during client recognition and loading.



Figure 6. Residue centrality profiles of Cdc37 and Cdk4 in the Hsp90-Cdc37-Cdk4 complex. Residue-based centrality distributions of Cdc37 (A) and Cdk4 (B) are shown in domain-colored bars. The Cdc37-NTD (residues 1-147) is in blue bars, Cdc37-M/C (residues 148-260) in red bars. The Cdk4-N lobe (residues 1-99) is in blue bars, and Cdk4 C-lobe (residues 100-295) is in red bars. (C) Structural mapping of high centrality residues in the Hsp90-Cdc37-Cdk4 complex. For clarity, only Hsp90-B (pink ribbons) is shown. Cdc37 in red ribbons, Cdk4 in yellow ribbons. (D) A close-up of high centrality residues at the Hsp90-B/Cdc37-NTD interface. The Cdc37-NTD residues (pS13, D14, D15, E16, E18, H20, P21, N22, I23, F29, W31, and R33) are highlighted in red spheres. The Hsp90-B interfacial residues (K399, K402, R405, and K406) and adjacent hydrophobic sites (I370, F376, and Y373) are shown in pink spheres. 24 ACS Paragon Plus Environment

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Asymmetric Topography of Intermolecular Communities Reveals Functional Role of Cdc37 in Protecting Kinase Client in the Repressed State Recent experimental revelations indicated that in the absence of binding partners the closed Hsp90 state may be intrinsically asymmetric at the MD-CTD interface, whereas Cdc37 and client binding force Hsp90 to adopt a symmetric state.40 In this context, we examined organization and hierarchy of allosteric interaction networks in the chaperone-kinase complex. By using modular decomposition of the global interaction network, we quantified topography of allosteric interactions revealing an asymmetry of the community maps that may have functional relevance for the mechanism of kinase recruitment. Importantly, we found that high centrality residues may be assembled into local modules of strongly interacting nodes (Figure 7). Structural map of communities in the Hsp90-Cdc37-Cdk4 complex highlighted the prevalence of the intermolecular modules anchored by Cdc37-NTD residues and illustrated an asymmetric topography of mediating centers (Figure 7). The spatial distribution of the intermolecular communities displayed a clear bias towards the Hsp90-Cdc37-NTD and Cdc37-NTD/Cdk4-Clobe interfaces. We detected a densely populated network of the intermolecular communities at the Hsp90-MD interface with Cdc37-NTD (Figure 7B). Stable modules in this region are formed through an extensive network of electrostatic interactions and salt bridges: K399 (Hsp90)-D15 (Cdc37-NTD)-D18 (Cdc37-NTD), K402 (Hsp90)-R405 (Hsp90)-E16 (Cdc37-NTD), and N407 (Hsp90)-V403 (Hsp90)-V116 (Cdc37-NTD). The largest and most stable community in the Hsp90-Cdc37-NTD region is formed through interactions of the Hsp90 residues (L119, Q326, L327, and L395) and functional sites of Cdc37 (Y4, W7, I10) (Figure 7B). This broad community links phosphorylated sites on Cdc37 (Y4) with the energetic hotspots of Hsp90 stability and contributes decisively to the stability of the Hsp90-Cdc37 interface. 25 ACS Paragon Plus Environment


Figure 7. Structural map of major intermolecular communities. (A) An overview of the communities (in colored spheres), Hsp90-A monomer is shown in orange ribbons and Hsp90-B monomer is in pink ribbons. Cdc37 is in red, and Cdk4 is in yellow ribbons. The residues in major communities are shown in spheres colored according to domain annotation. (B) The communities linking Hsp90-B and Cdc37-NTD : K399(Hsp90)-D15(Cdc37)-D18(Cdc37) (in orange spheres), K402(Hsp90)-R405(Hsp90)-E16(Cdc37) (in blue spheres), N407(Hsp90)V403(Hsp90)-V116(Cdc37) (in salmon spheres), L327(Hsp90)-Q326(Hsp90)-L119(Cdc37)W7(Cdc37)-I400(Hsp90)-V403(Hsp90)-L3095(Hsp90)-P387(Hsp90)-Y4(Cdc37)-Q397(Hsp90)I10(Cdc37) (in hot pink spheres). The key residues in the largest community are annotated. (C) The communities linking Cdc37-NTD and Cdk4 C-lobe: H20(Cdc37)-I23(Cdc37)-L28(Cdc37)26 ACS Paragon Plus Environment

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F130(Cdk4)-L131(Cdk4)-F159(Cdk4)-I136(Cdk4)-W31(Cdc37)-L128(Cdk4) (in blue spheres), and N22(Cdc37)-Q123(Cdk4)-V154(Cdk4)-V148(Cdk4)-L120(Cdk4) (in salmon spheres). (D) A single largest community at the Cdc37-M/C interface with the kinase N-lobe. The contributing kinase residues (Y17, V37, E56, V57, L60, and L74) are shown in yellow spheres, and Cdc37M/C residues (W168, D169, I241, K242) are shown in red spheres. Note contribution of conserved regulatory kinase sites E56, L60, and L74 from the regulatory αC-helix.

The analysis of the Hsp90-Cdc37 communities provided an interesting and novel rationale to various functional and biochemical experiments on regulation of the Hsp90-Cdc37 interactions. It has been recognized that both Hsp90 and Cdc37 are delicately regulated by phosphorylation. In particular, Cdc37 can be phosphorylated on Y4 and Y298 by Yes kinase103 and on S13 position by CKII kinase104 that are both Hsp90 clients. Phosphomimetic mutations of these Cdc37 sites can lead to impaired Cdc37-kinase binding and prevent kinase recruitment to the Hsp90-Cdc37. In our analysis, Cdc37-Y4 phosphorylation site is embedded into the largest intermolecular community. As a result, mutations in this single position can simultaneously disrupt multiple interactions with community neighbors and severely impair the stability of the complex. In the Hsp90–Cdc37–Cdk4 structure, Cdc37-S13 is phosphorylated and contributes to the intercommunity connectivity in the complex. We observed that the interaction cluster formed by pS13 with H33 and R36 of the Cdc37-NTD can link the Hsp90-Cdc37 and Cdc37-NTD-Cdk4 communities. A mediating role of S13 in connecting multiple communities in this region can explain a severe detrimental effect of phosphomimetic mutants in this position on regulation of the Hsp90-Cdc37 interactions. The network-centric interpretation of functional significance of 27 ACS Paragon Plus Environment


these sites for Hsp90-Cdc37 regulation is based on their integrating role in large intermolecular communities. In network terms, modifications of these sites would simultaneously weaken numerous interactions, compromising modularity and interconnectivity of the global network and consequently undermining efficiency of allosteric signaling. The asymmetry of the interfacial communities is manifested in a dense network of local modules is anchored by Cdc37-NTD residues, while Cdc37-M/C regions can be only weakly coupled to the Hsp90 and Cdk4 binding partners. The principal stabilizing community at the Cdc37-NTD interface with Cdk4 linked together Cdc37-NTD residues (H20, I23, L28, and W31) and Cdk4C-lobe residues (G127, L128, F130, L131, I136, H138, and F159). Strikingly, this community connected the recognition Cdc37-NTD motif (HPN) with the regulatory HRD and DFG kinase motifs (H138, F159). Notably, H138 and F159 residues from Cdk4 belong to the regulatory Rspine (L60, L74, H138, F159, and D196) whose cooperative assembly controls kinase activation.105 Through dense interactions formed in this community Cdc37-NTD can protect a partially unfolded kinase conformation, while also constraining the R-spine in the inactive arrangement (Figure 7C). These findings provide a compelling rationale to the experimental observations that the Hsp90 chaperone can force kinase to remain in the inactive repressed state and only after dissociation clients can be activated.106-108 Another important component of the global network was formed at the interface between Hsp90-MD/CTD and unfolded kinase segment of the N-lobe. The intermolecular communities W598(Hsp90)-M606(Hsp90)R612(Hsp90)-E94(Cdk4) and L343(Hsp90)-F344(Hsp90)-L91(Cdk4)-V89(Cdk4) protect the unfolded kinase segment. Several small communities can be formed by the Cdc37-M/C residues, including F341(Hsp90)-E235(Cdc37)-Q236(Cdc37), Q208(Cdc37)-R166(Cdc37)-V72(Cdk4) and Y17(Cdc37)-K35(Cdk4)-D169(Cdc37)-V20(Cdk4). The most interesting finding was the 28 ACS Paragon Plus Environment

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identification of a single largest community at the Cdc37-M/C interface with the kinase N-lobe that linked kinase residues (Y17, V37, E56, V57, L60, L74, A79) with Cdc37-M/C residues (W168, D169, I241, K242). Of particular significance is evidence that conserved regulatory kinase sites, such as catalytic residue E56 (from catalytic salt bridge pair K35-E56) and R-spine residues L60, L74 from the regulatory αC-helix (Figure 7D). As a result, Cdc37-M/C domain can aid in protecting the inactive R-spine arrangement of the kinase client. An important result of this analysis is that Hsp90-Cdc37 interfacial regions are coupled through communities with the key regulatory residues of Cdk4 that are evolutionary conserved and functionally essential across the spectrum of kinase clients. These kinase residues belong to the regulatory αC-helix (L60, L74, H138, and F159), the catalytic HRD motif (H138) and DFG motif (F159). Notably, these regulatory residues are known to control a dynamic kinase.105 Consequently, Hsp90 and Cdc37 can act cooperatively at the binding interfaces to shield unstable kinase client and simultaneously control positions of the regulatory kinase residues responsible for Cdk4 activation. We argue that these interactions may underlie a mechanism by which how Hsp90 and Cdc37 can restrain undesirable oncogenic activity of the kinase clients. Our results suggested that preservation of these binding interactions and corresponding communities may have evolutionary benefit for efficient recognition and processing of kinase clientele by the chaperone system. To summarize, the asymmetric topography of the intermolecular communities in the chaperone-kinase complex supported the indispensable mediating role of Cdc37 in allosteric regulation, particularly revealing specific contributions of Cdc37 domains in protecting unstable kinase client in the inactive dormant state. The observed modularity and asymmetry of the intermolecular communities may be an important global signature of the Hsp90-client interactions that enables efficient allosteric signaling in the complex required for productive kinase processing. 29 ACS Paragon Plus Environment


Modeling of Allosteric Communication Pathways: Communication Hotspots Define Regulatory Switch Points of the Hsp90-Cdc37 Interactions with Kinase Client Using the results of community decomposition, we explored edge betweenness (or edge centrality) in the global interaction network as a proxy for modeling of allosteric communication pathways. This parameter is defined as the ratio of all the shortest paths passing through a particular edge to the total number of shortest paths in the network. Accordingly, edges that interconnect many local communities (inter-modular bridges) would tend to have the higher edge betweenness values as compared to the intra-modular edges. We evaluated the edge centrality distribution based on all short inter-residue paths in the Hsp90-Cdc37-Cdk4 complex (Figure 8A). The distribution featured a sharp decline and long tail indicative of a small-world organization in which only a small number of edges have a significantly higher betweenness values as compared to the average. By considering the ensemble of short paths that connected the Cdc37-NTD/Cdk4 C-lobe interface with the ATP binding site, we detected a pronounced shift in the distribution towards high edge centrality values (Figure 8A). Only a moderate shift in the edge centrality was observed for the ensemble of short pathways connecting the kinase lobes (Figure 8A). Accordingly, the ensemble of signaling routes that pass through Cdc37-NTD interface may be important for coordinating allosteric communications in the chaperone complex. By considering short paths going through the high centrality edges, we also found that the Hsp90 and Cdc37-NTD regions harbor most of these communication hubs (Figure 8B). Strikingly, the inter-community bridges are often formed by phosphorylation and lysine acetylation sites, suggesting the previously unrecognized role of PTMs as potential transmitters of allosteric communications in the chaperone-client complexes.


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Figure 8. The distribution of edge centrality in the Hsp90-Cdc37-Cdk4 complex. (A)The edge centrality distribution is based on all short inter-residue paths in the Hsp90-Cdc37-Cdk4 complex (red bars). The edge centrality distribution for the ensemble of short pathways connecting the kinase lobes blue bars). The edge centrality distribution for the ensemble of short paths connecting the Cdc37-NTD/Cdk4 C-lobe interfacial residues with the ATP binding site (in green bars). (B) The domain-based distribution of high centrality inter-community edges (red bars). (C) Structural mapping of the major communities in the Hsp90 dimer (shown in green spheres for Hsp90-A monomer and cyan spheres for Hsp90-B monomer. The positions of important conserved PTM sites in the human Hsp90β are highlighted in red spheres and annotated. Note that these PTM sites can often assume role of the inter-community bridges. 31 ACS Paragon Plus Environment


To highlight this finding, we mapped major communities in the unbound Hsp90 dimer along with positions of conserved PTM sites (Figure 8C). Notably, the PTM sites are distributed along the community edges or are located at the inter-community boundaries. These observations suggested that inter-domain communication pathways in the chaperone-kinase complex may exploit these regulatory sites for efficient modulation of allosteric signals.

Using a community-hopping model of allosteric pathways80 we reconstructed ensemble of allosteric communication pathways that pass through high centrality edges (Figure 9A). The topography of this map revealed major highways absorbing most of the communication ‘traffic’ between the ATP binding sites of Hsp90 and kinase client (Figure 9B). By “zooming in” on the residue pairs with the highest edge betweenness, we identified key interactions that enable allosteric communications in the Hsp90-Cdc37-Cdk4 complex. The inter-modular edges of highest centrality included M363-S365, K350-S365, K399 (Hsp90-MD)-D15 (Cdc37-NTD), and K399 (Hsp90-MD)-E18 (Cdc37-NTD) connections (Figure 9B). Among communication hotspots that coordinate cross-talk between binding interfaces are Cdc37-NTD residues S13, F29, and W31. These sites are involved in high centrality bridges with Hsp90-MD (S13) and the regulatory DFG motif of Cdk4 (F29, W31). Our model suggested that these communication hotspots may function as important network bridges or switches that coordinate allosteric interactions and signal transmission in the chaperone complex. Strikingly, many high centrality edges connected experimentally known points of allosteric regulation in the Hsp90. Furthermore, some of the key allosteric hubs in the complex corresponded to conserved PTM sites of the Hsp90 chaperone.


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Figure 9. Structural map of major communication pathways passing through high centrality edges in the Hsp90-Cdc37-Cdk4 complex. (A) A general overview and topography of communication pathways. Residues along these pathways are shown in green and cyan spheres for Hsp90 A and B monomers respectively. The positions of PTM sites in the Hsp90 chaperone are highlighted by red spheres. (B) A close-up of high centrality inter-modular connections is shown in domain-colored spheres. The functional sites of post-translational modifications in this communication map are highlighted in blue spheres and annotated.



It has been recognized that phosphorylation of the Hsp90 permits regulation of the conformational cycle by targeting spatially remote switch points of the inter-domain communication and these changes affect client maturation and the interaction with cochaperones.30 Our results provided a rationale of the experimentally observed effects by showing that global communication traffic may depend on the inter-modular bridges formed by PTM sites. In particular, phosphorylation of S13 in Cdc37-NTD is necessary for Cdc37 interaction with Hsp90 and also for chaperoning of numerous kinase clients including Cdk4. Consistent with these experiments, our results showed that the interactions formed by pS13 with H33 and R36 of the Cdc37-NTD form high centrality bridges connecting Hsp90-Cdc37 and Cdc37-Cdk4 communities. The emergence of high centrality edges near Cdc37-NTD binding interfaces further strengthened our arguments that Cdc37-NTD may be central in kinase recognition. Recent experiments identified residue S365 as an isoform-specific phosphorylation site in human Hsp90β chaperone.109 Mutations in this position may drastically affect client maturation and is believed to impair progression of the Hsp90-kinase cycle by altering conformational equilibrium and disfavoring the closed form of the Hsp90 dimer. Mutational analysis of K294 in human Hsp90α (K286 in human Hsp90β) demonstrated that its acetylation status is a strong determinant of client and cochaperone binding, since Hsp90 variants that cannot be acetylated at this position could impair chaperone function.110 These experimental studies attributed regulatory role of this site, located at the juncture between the charged linker and Hsp90-MD, to the local dynamic changes and loss of the intramolecular interactions caused by proximity to residues T285 and P287 from a hydrophobic cluster at the NTD-MD interface.


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Our results showed that the two key inter-modular bridges involve S365 site (M363-S365 and K350-S365) and may serve as central mediators of long-range communications in the complex (Figure 9B). Several other PTM sites, including K286, K319, K350 and K399, also contribute to the highest centrality edges (Figure 9B). These findings indicated that regulatory PTM sites can be interconnected along main communication pathways, assuming role of primary control points and gate-keepers of allosteric signaling in the chaperone-kinase complex. According to our results, the detrimental functional effect of mutations in these PTM sites can arise from global changes in the interaction network and alteration of primary communication routes in the system. In this novel interpretation of the experimental data, mutations of PTM sites may not necessarily signal structural changes or disfavor the closed form of Hsp90 dimer, but rather lead to rewiring of major communication pathways and consequently compromise efficiency of allosteric signaling in the system. Based on our results, we propose that several other phosphorylation and lysine acetylation PTM sites (K350, S391, K399, S452) emerged as major control points of longrange communications (Figure 9B) could also serve as potential allosteric switches of the Hsp90 interactions with kinase clients.

To determine specific role of communication hotspots in an allosteric cross-talk between binding interfaces, we generated ensembles of allosteric communication pathways connecting spatially separated functional sites in the Hsp90 and Cdk4 client. We applied a community-hopping model of allosteric pathways80 in which a pair of residues may be viewed as ‘source’ and ‘sink’ of the pathway. 100 suboptimal paths were computed for each of the studied residue connections. We generated the ensemble of short length pathways that connected the catalytic site (R392) in the Hsp90-NTD with functional sites in Cdk4 client kinase. One of the optimal pathways linked 35 ACS Paragon Plus Environment


Hsp90-R392 site with phosphorylation site T172 in C-lobe of Cdk4 client (Figure 10A). The optimal pathway proceeded from the catalytic R392 residue through (L395-Q397-I400-V403) and (L388-I362-R392) communities, reaching to the inter-modular edge formed by M363 and S365 residues, and connecting to the interfacial hotspot K399. This route traversed through the Hsp90/Cdc37-NTD interfacial bridge to reach to the interfacial hotspots of Cdc37-NTD (W31, F29, D14, and E18), and then linked via the regulatory DFG motif of Cdk4 (F159) to the phosphorylation site T172 of Cdk4 (Figure 10A). We also modeled communication pathways that link R392 site in the Hsp90-NTD with the Cdk4 N-lobe. In this case, optimal routes passed through Hsp90 community (R392-L388-I362) and navigated via high centrality edges connecting I362, M363, S365, K350 and K348 residues (Figure 10A). By traversing through mediating centers of Hsp90-MD, the paths reached the Hsp90 Src-loop residues (E344 and L343) and connected to the unfolded region of the kinase N-lobe (I87, V89, and L91). Significantly, in both cases, the ensembles of communication pathways showed a strong tendency to proceed through evolutionary conserved regulatory PTM sites.

In another series of computational experiments, we generated the ensemble of communication pathways that connect functional regions in Cdc37-NTD with the Cdc37 N-lobe (Figure 9B). Two equally probable alternative routes emerged, where the first path connected Cdc37-NTD residues (F29, W31) with the DFG motif in the C-lobe of Cdk4 and interfacial hotspots of Hsp90-CTD (W598), reaching out to the regulatory αC-helix of Cdk4 (T53, E67) (Figure 9B). The alternative route proceeded high centrality edges that connect communities near another regulatory center in the Hsp90-MD (F344, F433). Hence, both optimal pathways utilized structurally stable allosteric hotspots to efficiently transmit signal between binding interfaces. 36 ACS Paragon Plus Environment

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Figure 10. Modeling of allosteric communication pathways in the Hsp90-Cdc37-Cdk4 complex. (A) Representative optimal short paths from the ensemble of communication pathways connecting catalytic residue R392 (Hsp90-B NTD) with functional sites in Cdk4 client kinase. (B) The most probable pathways from the ensemble of short inter-residue routes connecting Cdc37-NTD functional sites with Cdk4 N-lobe regions. The most probable paths are shown and the respective residues are shown in domain-colored spheres and annotated.



Atomistic reconstruction of allosteric pathways revealed a well-defined topography of communication traffic in the complex that enables a cross-talk between regulatory residues and binding interfaces, where Cdc37-NTD regions play a central integral role. We found that Cdc37NTD residues form major mediating bridges between communities and dominate allosteric communications in the complex. Interestingly, optimal routes that interconnect spatially separated binding interfaces do not include the interfacial Cdc37-M/C residues, supporting the notion that C-terminal domain of Cdc37 may not be play a secondary role in an allosteric crosstalk during client recruitment. This model of client recognition and processing is reminiscent of a molecular mechanism underlying client binding by other chaperones studied by NMR experiments.111 our analysis also suggested that the regulatory role of allosteric control points in Hsp90 and Cdc37 may be determined by their role in the inter-community bridges of the global interaction network. The results rationalize the NMR experiments of the Hsp90-Cdc37 binding with protein kinase clients36,

37

and provide novel insights by revealing atomistic view of

allosteric signaling through a privileged group of functional centers.

Conclusions

In this study, we attempted to examine several outstanding questions concerning regulation of the Hsp90-Cdc37 interactions with client proteins by using an array of computational methods. The methodological framework of our study is based on integration of dynamic and networkcentric approaches into a unified and robust computational strategy for quantifying mechanisms of allosteric regulation. Using these approaches, we characterized allosteric interaction networks and hierarchy of the intermolecular binding interfaces in the Hsp90-Cdc37-client complexes Our 38 ACS Paragon Plus Environment

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results demonstrated that allosteric regulation of the Hs90 binding may couple the intra-domain and inter-domain interactions in a complex and dynamic interaction network. Through systematic identification of stabilization centers and centrality sites in the interaction networks, we characterized allosteric cross-talk and hierarchy of the intermolecular binding interfaces in the Hsp90-Cdc37-client complex. According to our findings, Cdc37-NTD can mediate kinase recognition by orchestrating a cascade of allosteric interactions with the Hsp90 chaperone and Cdk4 client. The results also quantified functional specialization of the Hsp90 and Cdc37 domains in protecting unstable kinase client in its repressed inactive form. Modeling of allosteric pathways in the chaperone complex has further clarified structural and energetic signatures of allosteric hotspots, particularly linking PTM sites with their important role in allosteric interactions. By identifying principal communities and reconstructing communication pathways, we obtained novel molecular insights about specific functional roles of key PTM sites serving as control points of allosteric signaling. The emerged interconnectivity and coupling of regulatory sites in the Hsp90 chaperone may be useful in expanding array of therapeutic strategies for design of novel allosteric inhibitors targeting allosteric switch points that control Hsp90 interactions with oncogenic kinase clients.



AUTHOR INFORMATION * Corresponding Author Phone: 714-516-4586 Fax: 714-532-6048 E-mail: [email protected]

Acknowledgment This work was partly supported by institutional funding from Chapman University.

ABBREVIATIONS 90-kilodalton Heat shock protein, Hsp90; human protein Hsp90, N-terminal domain of Hsp90, Hsp90-NTD; middle domain of Hsp90, Hsp90-MD; C-terminal domain of Hsp90, Hsp90-CTD; N-terminal domain of Cdc37, Cdc37-NTD; middle and C-terminal domain of Cdc37, Cdc37M/C; hydrogen/deuterium exchange mass spectrometry, HX-MS; molecular dynamics, MD; discrete molecular dynamics, DMD; flexibility-rigidity index, FRI; mutual Information, MI; multiple sequence alignment, MSA; mutual information server to infer coevolution, MISTIC.


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Functional Role and Hierarchy of the Binding Interfaces in Recruitment of Protein Kinase Clients to the Hsp90-Cdc37 Chaperone Machine: Structure-Based Network Analysis of Allosteric Interactions and Communication Pathways

Gabrielle Stetz, Gennady M. Verkhivker

For Table of Contents Use Only


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Figure 1. Structural mapping and analysis of the binding interfaces in the Hsp90-Cdc37-Cdk4 complex. The overview of the global topology and intermolecular interfaces in the Hsp90-Cdc37-Cdk4 complex is shown in ribbons (A) and surface representations (B). Hsp90-A monomer is shown in orange ribbons and Hsp90-B monomer B is in pink ribbons. Cdc37 is in red ribbons, and Cdk4 kinase is in yellow ribbons. Each monomer in the full length human Hsp90β protein is divided into three domains: NTD (residues 1-215), MD (residues 216-552), and CTD (residues 553-690). The domain annotation is based on comprehensive sequence analysis of the Hsp90 family.89,90 Cdc37 domains are annotated as follows: Cdc37-NTD (residues 1-147), and Cdc37-M/C (residues 148-260). The Cdk4 N-lobe (residues 1-99) and Cdk4 C-lobe (residues 100-295) annotations are consistent with the original study.39 (C) The Hsp90-Cdc37-NTD intermolecular cluster is formed by phosphorylated S13 (Cdc37-NTD) interacting with Cdc37-NTD residues (H33, R36) and Hsp90K406. Another binding interface is mediated by a network of salt bridges formed by the Cdc37-NTD residues (D14, D15, E16, D17, and E18) and Hsp90-MD residues (K399, K402, R405, and K406). (D) The Hsp90-Cdk4 interface involves the Src-loop region in the Hsp90-MD (residues F341, L343, F344, E345) and Hsp90-CTD residues L611, R612 and M602. The residues are shown in domain-colored spheres. 133x99mm (300 x 300 DPI)

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Figure 2. FRI profiles of the Hsp90 dimer. Residue-based FRI distributions of the Hsp90 dimer (A), Cdc37 (B) and Cdk4 kinase client (C) in the unbound form (shown in green lines) and in the Hsp90-Cdc37-Cdk4 complex (shown in maroon lines). (D) Structural mapping of the inter-domain cluster of rigid residues from Hsp90-MD (P287, W289, Y356, V360 and Hsp90-NTD (F208). Hsp90-A residues are shown in orange spheres and Hsp90-B monomer residues are in pink spheres. The cluster is linked to catalytic residue R392 and ATP (shown in atom-colored spheres). (E) Structural map of stable hydrophobic residues (shown in pink spheres) forming an interacting cluster in the Hsp90-MD (L369-I370-V381-L401-I404). These hydrophobic residues are proximal to a group of lysine residues K399, K402, R405, K406, and K411 (shown in hot pink spheres) that form specific interactions with Cdc37-NTD residues (shown in red spheres). (F) Structural map of stable residues at the interface of Hsp90-CTD (R338, L343, F344, N346, and M602 shown in pink spheres) and unstructured segment of the kinase N-lobe (F92, V93, E94, V96 shown in yellow spheres). These residues are flexible in the unbound proteins but become rigidified in the chaperone-kinase complex. 133x99mm (300 x 300 DPI)


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Figure 3. Alanine scanning and protein stability analysis of the Hsp90-Cdc37-Cdk4 complex. Protein stability changes are computed for the Hsp90 dimer (A, B), Cdc37 (C) and Cdk4 (D) using a systematic alanine scanning of the protein residues to alanine and computing the effect of each mutation on protein stability with the FoldX approach. The profiles computed for the unbound Hsp90 dimer, Cdc37 and native Cdk4 kinase structure are shown in brown filled bars. The stability changes for protein residues in the Hsp90Cdc37-Cdk4 complex are shown in domain-colored bars. The Hsp90-NTD residues are shown in red bars, Hsp90-MD residues are in blue bars, and Hsp90-CTD residues are in green bars. The Cdc37-NTD residues are in red bars, Cdc37-M/C residues are in blue bars. The Cdk4-N lobe residues are in red bars, and Cdk4 C-lobe residues are in blue bars. 133x99mm (300 x 300 DPI)



Figure 4. Structural mapping of protein stability hotspot clusters. (A) The overview of the intermolecular interfaces and energetic hotspot centers in the Hsp90-Cdc37-Cdk4 complex. Hsp90-A monomer is shown in orange ribbons and Hsp90-B monomer B is in pink ribbons. Cdc37 is in red ribbons, and Cdk4 kinase is in yellow ribbons. The location of three major centers of energetic hotspots is indicated by rectangular boxes colored in red (with a close-up in panel B), blue (a close-up is presented in panel C), and green (a close-up is in panel D). (B) Structural mapping of the intramolecular cluster connecting energetic hotspots near the NTD-MD interface. For clarity only Hsp90-B monomer (colored in pink ribbons) is shown. The high stability clusters I288-F304-L332 and P287-W289-F304-Y356 are shown in domain-colored spheres. These residues are directly linked to L388, I390 and R392 (in sticks) that bridge NTD and MD regions near the catalytic site. Catalytic residue R392 and ATP are shown in atom-colored spheres. (C) A close-up of the protein stability hotspot formed by the hydrophobic cluster (I404-V381-I408-L369-I370-L401). The hotspot residues are shown in domain-colored pink spheres (Hsp90-B monomer of the dimer). This stability hotspot is immediately proximal to the Hsp90 binding residues K399, K402, R405, K406 (shown in pink sticks ) that form a network of salt bridges with Cdc37-NTD residues D14,D15, E16,D17,E18 ( in red sticks). Hsp90-B in pink ribbons, Cdc37 in red ribbons, and Cdk4 is in yellow ribbons. (D) The protein stability hotspot cluster F376-L343-F433-N436 (in pink spheres) anchors the Src-loop residues R338, F341, L343, F344 (in sticks) that make contacts with the Cdk4 N-lobe regions. The Cdc37-M/C domain is shown in red ribbons and Cdk4N lobe is shown in yellow ribbons.

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Figure 5. Residue centrality profiles of the Hsp90 dimer. Residue-based centrality distributions of the Hsp90 monomers in the chaperone-kinase complex (A, B). The client-induced differential changes in the residue centrality of the Hsp90 residues (C, D) are obtained from differences between residue betweenness values in the complex and unbound Hsp90 dimer. The distributions are derived by averaging computations of network parameters over equilibrium ensembles extracted from DMD trajectories. The distributions are shown in domain-colored bars. The Hsp90-NTD residues are shown in red bars, Hsp90-MD residues are in blue bars, and Hsp90-CTD residues are in green bars. 133x99mm (300 x 300 DPI)



Figure 6. Residue centrality profiles of Cdc37 and Cdk4 in the Hsp90-Cdc37-Cdk4 complex. Residue-based centrality distributions of Cdc37 (A) and Cdk4 (B) are shown in domain-colored bars. The Cdc37-NTD (residues 1-147) is in blue bars, Cdc37-M/C (residues 148-260) in red bars. The Cdk4-N lobe (residues 199) is in blue bars, and Cdk4 C-lobe (residues 100-295) is in red bars. (C) Structural mapping of high centrality residues in the Hsp90-Cdc37-Cdk4 complex. For clarity, only Hsp90-B (pink ribbons) is shown. Cdc37 in red ribbons, Cdk4 in yellow ribbons. (D) A close-up of high centrality residues at the Hsp90B/Cdc37-NTD interface. The Cdc37-NTD residues (pS13, D14, D15, E16, E18, H20, P21, N22, I23, F29, W31, and R33) are highlighted in red spheres. The Hsp90-B interfacial residues (K399, K402, R405, and K406) and adjacent hydrophobic sites (I370, F376, and Y373) are shown in pink spheres. 133x99mm (300 x 300 DPI)


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Figure 7. Structural map of major intermolecular communities. (A) An overview of the communities (in colored spheres), Hsp90-A monomer is shown in orange ribbons and Hsp90-B monomer is in pink ribbons. Cdc37 is in red, and Cdk4 is in yellow ribbons. The residues in major communities are shown in spheres colored according to domain annotation. (B) The communities linking Hsp90-B and Cdc37-NTD : K399(Hsp90)-D15(Cdc37)-D18(Cdc37) (in orange spheres), K402(Hsp90)-R405(Hsp90)-E16(Cdc37) (in blue spheres), N407(Hsp90)-V403(Hsp90)-V116(Cdc37) (in salmon spheres), L327(Hsp90)-Q326(Hsp90)L119(Cdc37)-W7(Cdc37)-I400(Hsp90)-V403(Hsp90)-L3095(Hsp90)-P387(Hsp90)-Y4(Cdc37)-Q397(Hsp90)I10(Cdc37) (in hot pink spheres). The key residues in the largest community are annotated. (C) The communities linking Cdc37-NTD and Cdk4 C-lobe: H20(Cdc37)-I23(Cdc37)-L28(Cdc37)-F130(Cdk4)L131(Cdk4)-F159(Cdk4)-I136(Cdk4)-W31(Cdc37)-L128(Cdk4) (in blue spheres), and N22(Cdc37)Q123(Cdk4)-V154(Cdk4)-V148(Cdk4)-L120(Cdk4) (in salmon spheres). (D) A single largest community at the Cdc37-M/C interface with the kinase N-lobe. The contributing kinase residues (Y17, V37, E56, V57, L60, and L74) are shown in yellow spheres, and Cdc37-M/C residues (W168, D169, I241, K242) are shown in red spheres. Note contribution of conserved regulatory kinase sites E56, L60, and L74 from the regulatory αChelix. 133x99mm (300 x 300 DPI)



Figure 8. The distribution of edge centrality in the Hsp90-Cdc37-Cdk4 complex. (A)The edge centrality distribution is based on all short inter-residue paths in the Hsp90-Cdc37-Cdk4 complex (red bars). The edge centrality distribution for the ensemble of short pathways connecting the kinase lobes blue bars). The edge centrality distribution for the ensemble of short paths connecting the Cdc37-NTD/Cdk4 C-lobe interfacial residues with the ATP binding site (in green bars). (B) The domain-based distribution of high centrality intercommunity edges (red bars). (C) Structural mapping of the major communities in the Hsp90 dimer (shown in green spheres for Hsp90-A monomer and cyan spheres for Hsp90-B monomer. The positions of important conserved PTM sites in the human Hsp90β are highlighted in red spheres and annotated. Note that these PTM sites can often assume role of the inter-community bridges. 133x99mm (300 x 300 DPI)


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Figure 9. Structural map of major communication pathways passing through high centrality edges in the Hsp90-Cdc37-Cdk4 complex. (A) A general overview and topography of communication pathways. Residues along these pathways are shown in green and cyan spheres for Hsp90 A and B monomers respectively. The positions of PTM sites in the Hsp90 chaperone are highlighted by red spheres. (B) A close-up of high centrality inter-modular connections is shown in domain-colored spheres. The functional sites of posttranslational modifications in this communication map are highlighted in blue spheres and annotated. 133x99mm (300 x 300 DPI)



Figure 10. Modeling of allosteric communication pathways in the Hsp90-Cdc37-Cdk4 complex. (A) Representative optimal short paths from the ensemble of communication pathways connecting catalytic residue R392 (Hsp90-B NTD) with functional sites in Cdk4 client kinase. (B) The most probable pathways from the ensemble of short inter-residue routes connecting Cdc37-NTD functional sites with Cdk4 N-lobe regions. The most probable paths are shown and the respective residues are shown in domain-colored spheres and annotated.

133x99mm (300 x 300 DPI)


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Functional Role and Hierarchy of the Binding Interfaces in Recruitment of Protein Kinase Clients to the Hsp90-Cdc37 Chaperone Machine: Structure-Based Network Analysis of Allosteric Interactions and Communication Pathways

Gabrielle Stetz, Gennady M. Verkhivker

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88x35mm (300 x 300 DPI)


Functional Role and Hierarchy of the Intermolecular Interactions in

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