Molecular Dynamics Simulations and Structural Analysis of Giardia

Article pubs.acs.org/jcim

Molecular Dynamics Simulations and Structural Analysis of Giardia duodenalis 14-3‑3 Protein−Protein Interactions Ylenia Cau,†,∇ Annarita Fiorillo,‡,∇ Mattia Mori,†,§ Andrea Ilari,∥ Maurizo Botta,*,†,# and Marco Lalle*,⊥ †

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, via Aldo Moro 2, 53019 Siena, Italy Dipartimento di Scienze Biochimiche, Sapienza Università di Roma, Piazzale A. Moro 5, 00185 Roma, Italy § Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Roma, Italy ∥ CNR-Institute of Molecular Biology and Pathology (IBPM), c/o Department Biochemical Sciences “A. Rossi Fanelli”, University Sapienza, P.le A. Moro 5, 00185 Roma, Italy # Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Suite 333, 1900 North 12th Street, Philadelphia, Pennsylvania 19122, United States ⊥ Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy ‡

S Supporting Information *

ABSTRACT: Giardiasis is a gastrointestinal diarrheal illness caused by the protozoan parasite Giardia duodenalis, which affects annually over 200 million people worldwide. The limited antigiardial drug arsenal and the emergence of clinical cases refractory to standard treatments dictate the need for new chemotherapeutics. The 14-3-3 family of regulatory proteins, extensively involved in protein−protein interactions (PPIs) with pSer/pThr clients, represents a highly promising target. Despite homology with human counterparts, the single 14-3-3 of G. duodenalis (g14-3-3) is characterized by a constitutive phosphorylation in a region critical for target binding, thus affecting the function and the conformation of g14-33/clients interaction. However, to approach the design of specific small molecule modulators of g14-3-3 PPIs, structural elucidations are required. Here, we present a detailed computational and crystallographic study exploring the implications of g143-3 phosphorylation on protein structure and target binding. Self-Guided Langevin Dynamics and classical molecular dynamics simulations show that phosphorylation affects locally and globally g14-3-3 conformation, inducing a structural rearrangement more suitable for target binding. Profitable features for g14-3-3/clients interaction were highlighted using a hydrophobicity-based descriptor to characterize g14-3-3 client peptides. Finally, the X-ray structure of g14-3-3 in complex with a mode-1 prototype phosphopeptide was solved and combined with structure-based simulations to identify molecular features relevant for clients binding to g14-3-3. The data presented herein provide a further and structural understanding of g14-3-3 features and set the basis for drug design studies.

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INTRODUCTION Giardia duodenalis (syn. lamblia or intestinalis) is a worldwide protozoan parasite that colonizes the upper portions of the small intestine of mammals, including humans, causing giardiasis, the most common parasitic gastrointestinal diarrheal disease worldwide.1 More than 200 million symptomatic human cases are annually reported, mainly in developing countries although giardiasis is a relevant public health concern also in industrialized countries where it has caused large outbreaks.2 Infection is acquired by ingestion of cysts, the environment resistant stage, through consumption of contaminated water and food. Symptoms are caused by the actively replicating trophozoites, which attach to the surface of enterocytes without penetration of the epithelium or invasion of the surrounding tissues. A broad spectrum of clinical manifestation is associated with giardiasis ranging from lack of © XXXX American Chemical Society

symptoms to acute and chronic diarrhea. In malnourished children, the infection has been linked to impaired growth and cognitive functions, and long-term consequences including extraintestinal manifestation and postinfectious irritable bowel syndrome have been also associated with giardiasis.1 No human vaccines are yet available, and treatment of symptomatic cases relies on a limited panel of effective drugs, such as metronidazole. However, relevant side effects associated with these compounds and the emergence of treatment-refractory cases (10−20%) pose a serious risk for the future use of these drugs and highlight the urgent need of novel antigiardial treatment options.3 Received: July 20, 2015

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DOI: 10.1021/acs.jcim.5b00452 J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

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Journal of Chemical Information and Modeling In the last years, in silico methods coupled with experimental work have led to the concrete possibility of developing pharmacological interventions against cardiovascular, HIV, viral, and cancer diseases, with particular emphasis on the design of small drug-like molecules that modulate specific protein−protein interactions (PPIs).4−17 Among druggable PPIs, 14-3-3/clients interactions are a paradigmatic case. Misregulation of 14-3-3 genes has been linked to various human pathologies such as cancer and neurodegenerative disorders, justifying the increasing interest in 14-3-3s as therapeutic targets.18−20 14-3-3 is a family of dimeric phosphoserine/phosphothreonine (pSer/pThr) binding proteins, highly conserved in all eukaryotics, which directly interact with hundreds of client proteins thus exerting a crucial role in the regulation of a wide range of cellular processes (e.g., metabolism, cell cycle, protein trafficking). 14-3-3s preferentially bind to well conserved pSer/pThr-containing motifs, namely mode-1 [RSX(pS/pT)XP], mode-2 [RX(Y/F)X(pS/ pT)XP], and mode-3 [X(pS/pT)X1−2-COOH],21,22 even though cases of phosphorylation-independent binding have been also reported.23 From a structural standpoint, each 14-3-3 monomer consists of nine antiparallel α-helices (α1-α9) able to dimerize via the N-terminus (α1-α4), assuming a characteristic W-like folding.24 Four α-helices (α3, α5, α7, and α9) form an amphipathic binding groove, consisting of a cluster of highly conserved positively charged residues on one side and a hydrophobic patch on the other, which accommodates and directly contacts clients proteins or phosphopeptides. A single 14-3-3 isoform (g14-3-3) is present in G. duodenalis.25,26 Unique among all known 14-3-3s, two posttranslational modifications (PTMs) have been observed in g143-3, namely the constitutive phosphorylation of threonine 214 (Thr214, in the α8-α9 loop) and polyglycylation, the addition of a polyglycine chain to the lateral chain of glutamic acid 246 at the C-terminus. Both PTMs are essential for in vivo g14-3-3 functionality.27,28 Recently, we have solved the crystal structure of unmodified g14-3-3 in the apo form, showing that it possesses an uncommon “open” conformational arrangement.29 The use of the phosphorylation-mimetic T214E and the nonphosphorylatable T214A mutants, combined with preliminary in silico studies, indicated that Thr214 phosphorylation has a relevant impact on 14-3-3 binding activity, at both structural and functional levels, thus affecting the parasite life cycle by impairing cyst development.27,29 This evidence led us to hypothesize that Thr214 phosphorylation may act as a “lock” in the C-terminal region, which promotes a conformational restraint and increases the efficiency of g14-3-3 binding to a number of client proteins.29 Based on the key role of g14-3-3 phosphorylation in affecting the function and the conformation of g14-3-3/clients interaction, structural elucidations are required before to approach the design of specific small molecule modulators of g14-3-3 PPIs. Here, we present a detailed computational and crystallographic study on the structural implications of g14-3-3 phosphorylation at Thr214. Self-Guided Langevin Dynamics (SGLD) simulations on representative tripeptides were performed to explore efficiently the local conformational space of g14-3-3 within the phosphorylation site at Thr214. Classical molecular dynamics (MD) simulations were further used to investigate the global effect of phosphorylation on dimeric full-length g14-3-3, as well as the binding properties of a number of representative client phosphopeptides. A hydrophobicity-based descriptor was further used to characterize g14-

3-3 client peptides, thus highlighting profitable features for g143-3/clients interaction. Finally, the X-ray crystallographic structure of g14-3-3-polyG10 apo and in complex with a mode-1 prototype phosphopeptide was solved, supporting our theoretical observations.

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MATERIALS AND METHODS Chemicals. The soluble A8Ap phosphopeptide (ARAApSAPA) reproducing a mode-1 14-3-3 binding motif30 was synthesized by Primm srl, Italy. Proteins Expression and Purification. The GST-g14-3-3polyG10 expression vector has been described elsewhere.29 For recombinant protein expression in E. coli, bacteria were grown in SOB medium to OD600 = 0.6−0.8 and induced with 0.5 mM IPTG at 30 °C for 4 h. GST-g14-3-3-polyG10 was affinity purified on glutathione-sepharose 4B (GE Healthcare, Little Chalfont, UK) and released from GST by digestion with PreScission protease (GE Healthcare) at 4 °C for 16 h according to the manufacturer. Protein was dialyzed by PM-5 membrane o.n. at 4 °C in 50 mM Tris-HCl pH 7.5 and concentrated using Centricon 10 (Millipore Corporation, Bedford, MA, USA). Protein concentration was measured with Bradford’s method (BioRad, Hercules, CA, USA). Protein was stored at −70 °C until use. Protein Crystallization, Data Collection, and Processing. Crystals of g14-3-3-polyG10 were grown by the hanging drop vapor diffusion method at 293 K using a protein sample 0.4 mM. The crystals of g14-3-3-polyG10 in the apo form were obtained using a reservoir solution containing PEG 6000 10% w/v, lithium chloride 1 M, and HEPES 100 mM at pH 7.0. Crystals grew in 1 week with the following dimensions: 0.1 × 0.1 × 0.1 mm3. The g14-3-3-polyG10/A8Ap complex was obtained by mixing protein and peptide in a ratio of about 2:1. The final solution contained A8Ap 0.6 mM and g14-3-3polyG10 0.33 mM. The crystals of g14-3-3-polyG10 in complex with A8Ap were grown using a reservoir solution containing PEG 5000 MME 25% w/v, lithium acetate 0.2 M. Crystals, grew in 1 week with the following dimensions: 0.2 × 0.1 × 0.1 mm3. The crystals were cryo-protected with 20% PEG 400, mounted in nylon loops ,and flash-cooled by submersion into liquid N2 for transport to the synchrotron-radiation source. Crystals were poorly reproducible, and most of them diffracted at low resolution. X-ray diffraction data of g14-3-3-polyG10 in the apo form were collected at 4.0 Å resolution as 0.5° oscillation frames at 100 K on the beamline BL14-2 at Bessy (Berlin, Germany) using a MAR CCD detector. The data were processed and scaled using the suite HKL200031 and the programs of the CCP4 suite.32 X-ray diffraction data of g14-3-3polyG10 in complex with A8Ap were collected at 3.1 Å resolution as 0.5° oscillation frames at 100 K on the beamline BL14-1 at Bessy (Berlin, Germany) using a hybrid pixel detector Pilatus 6M. The data were processed using the program XDS33 and programs of the CCP4 suite.32 Data reduction and data scale statistics are reported in Table 1. The structures were solved by molecular replacement with the program Phaser34 using the structure of g14-3-3 as the search model (PDB code 4F7R).29 Model building was performed using COOT.35 The structure was refined using Refmac5.36 The refinement statistics are reported in Table 1. Structural figures were generated with PyMol.37 Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 5BY9 (g14-3-3-polyG10 in the apo form) and 4ZQ0 (g14-3-3-polyG10 in complex with A8Ap). B


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neutralize the whole charge, the required number of Na+ counterions was then added. All simulations were carried out using the AMBER12 package39 under periodic boundary conditions using a 2 fs time step and SHAKE algorithm43 to constrain all bonds between hydrogen and heavy atoms. A cutoff of 10 Å was applied to nonbonding interactions. Water molecules were minimized [500 steps with the steepest descendent algorithm (SD) and additional 500 steps with the conjugate gradient algorithm (CG)], while the protein was kept frozen. Then, the whole systems were minimized by means of 1500 steps of SD and 3000 steps CG. Systems were then heated at constant volume (NVT ensemble) up to 300 K for 80 ps using the Langevin thermostat44,45 and additionally equilibrated at constant pressure (NPT ensemble, Berendsen barostat) for 80 ps to achieve convergence of the density. As a production run, 200 ns of SGLD simulation was carried out on all tripeptides using a guiding temperature of 400 K, while 200 ns of classical MD at constant pressure and 300 K temperature was performed for other systems. Energy minimization and classical MD simulations were performed with the PMEMD module, while SANDER was used to run SGLD simulations. The particle-mesh Ewald procedure was used to handle longrange electrostatic interactions. In all MD simulations, the production of unbiased trajectories was performed within the NVT ensemble (constant volume), with the temperature being controlled by the Langevin thermostat. Four independent replicas for each system were simulated by MD. For the sake of clarity, only a representative simulation for each system is described herein, as all replicas provided very similar outcomes. RMSD, RMSF, DSSP, distance calculations, and clustering analysis were performed with the PTRAJ module of AmberTools13.46 Clustering analysis was performed using the average-linkage algorithm.47 Most populated clusters in pThr214-g14-3-3 and T214E-g14-3-3 simulation represented 62.5% and 75% of the total population respectively, while for the WT-g14-3-3 system four clusters with a total population of 18% were identified. The free energy of binding for g14-3-3/ peptide complexes was calculated using the MM-PBSA Python script implemented in Amber12.48 A more detailed explanation of the MM-PBSA theory, running equations, and application notes can be found in the Amber12 and AmberTools reference manuals (available at http://ambermd.org/doc12) and in Srinivasan et al.,49 in addition to a number of reviews and papers summarizing the contents and applications of the MMPBSA method.50−53 The Xmgrace program (http://plasma-gate.weizmann.ac.il/ Grace/) was used to plot two-dimensional plots of RMSD, RMSF, and distance. g14-3-3 residue numbering is according to g14-3-3 sequence, UniProt accession number Q2QBT8. For graphic representation, a representative structure was extracted from each MD trajectory. To this end, the frame endowed with the lowest RMSD variation with respect to the average structure of MD trajectories, calculated within frames at geometric convergence, was considered as representative. Calculation of Peptides Hydrophobicity. According to the Engelman scale,54 hydrophobicity of selected phosphopeptides was calculated as the sum of individual amino acid contributions. In detail, we considered sequences composed of 15 amino acids having the phosphorylated serine or threonine residue in the central position. Since the numeric contribution of phosphorylated residues is not available, the hydrophobicity of peptides was calculated in the nonphosphorylated form, assuming that the incremental contribution given by phosphor-

Table 1. Crystallographic Data Reduction, Processing, and Refinement g14-3-3-polyG10 PDB code space group unit cell dimensions

resolution range (Å) mean I/sigma(I) completeness (%) multiplicity Rmerge χ2/CC1/2 no. mon/ASU refinement statistics resolution range (Å) no. reflections R Rfree RMSD bond length RMSD bond angle residues in preferred region of Ramachandran plot (%) residues in allowed region of Ramachandran plot (%) a

g14-3-3-polyG10/A8Ap

5BY9 P32 a = 100.2 Å, b = 100.2 Å, c = 40.9 Å 40−4 (4.14−4.0) 5.13 (1.67) 92.5 (84.4) 2.0 (1.9) 0.154 (0.40) 1.21 (1.25) 4

4ZQ0 P1 21 1 a = 69.96 Å, b = 109.41 Å, c = 78.57 Å, β = 92.64

40.0−4.0 (4.1−4.0) 11755 (774) 0.23 (0.27) 0.23 (0.31) 0.0045 0.773 926 (100%)

48.0−3.10 (3.18−3.10) 20475 (1542) 0.20 (0.31) 0.27 (0.36) 0.005 0.988 886 (96%)

3 (0%)

41 (4%)

48.0−3.1 (3.31−3.10)a 10.3 (2.0) 99.9 (100) 5.2 (5.3) 0.145 (0.91) 99.7 (77) 4

Values given in parentheses refer to the highest resolution shell.

The B-factor is equal to 8π2⟨μ2⟩ where ⟨μ2⟩ is the mean squared displacement of the atoms. For apo g14-3-3-polyG10 (PDB code: 5BY9) the atomic average B-factor was 115 Å2 corresponding to a root mean deviation of 1.2 Å, whereas the peptide bound g14-3-3-polyG10 (PDB code: 4ZQ0) displays an atomic average B-factor of 80 Å2 corresponding to a root mean deviation of 1.0 Å. Finally, for the apo WT-g14-3-3 (PDB code: 4F7R) the atomic average B-factor was 63 Å 2 corresponding to a root mean deviation of 0.9 Å. MD Simulations. SGLD simulations were carried out on nine tripeptides (Gly-Thr-Gly, Ala-Thr-Ala, Leu-Thr-Glu, GlypThr-Gly, Ala-pThr-Ala, Leu-pThr-Glu, Gly-Glu-Gly, Ala-GluAla, Leu-Glu-Glu). Given the importance of 14-3-3 self-dimerization in phosphopeptide binding and biological activity,38 here classical MD simulations were carried out on unmodified wild type dimeric g14-3-3 (WT-g14-3-3, PDB ID: 4F7R),29 on dimeric g14-3-3 phosphorylated in silico on Thr214 (pThr214-g14-3-3), and on dimeric g14-3-3 with Thr214 mutated in silico to Glu (T214E-g14-3-3) as well as on dimeric pThr214-g14-3-3polyG10 in complex with A8Ap phosphopeptide core (ApSAP) and four 15-mer phosphopeptides, namely KSR S297 , CDC25CS216, KSRS392, and TSC2S1210 (Supporting Information, Table S1). The initial unstructured conformation of the tripeptides was modeled using the LEAP module of AMBER12,39 while 15-mer phosphopeptides were modeled using as the template the newly solved g14-3-3-polyG10 structure in complex with the A8Ap phosphopeptide core (PDB ID: 4ZQ0). The AMBER force field ff12SB,40 which includes revised backbone and side chain torsion parameters over ff99SB,40 was used for standard amino acids, whereas parameters for pSer/pThr optimized by Homeyer et al. were used.41 Each system was solvated with a 8 Å cuboid box of explicit TIP3P water molecules.42 To C


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Figure 1. Phi (Φ) and Psi (Ψ) angles distribution for (A) Gly-Thr-Gly, (B) Ala-Thr-Ala, and (C) Leu-Thr-Glu (upper panels), after Thr phosphorylation (middle panels) and after Thr to Glu mutation (lower panels). Map colors indicate the dihedral angles combination frequency according to the respectively color bars.

Boltzmann equation using the default parameters.55 Electrostatic surface potentials were then visualized with PyMol.37

ylation is the same in all peptides. On the basis of a preliminary benchmarking study performed on phosphopeptides with known relative affinity to g14-3-3,27 hydrophobicity values in the range of −17.9 to −34.1 were considered as suitable for binding to the amphipathic groove of g14-3-3. This range was furthermore used to check the g14-3-3 binding properties of phosphopeptides reproducing putative 14-3-3 binding sites present in g14-3-3 interactome.26 Measurement of Experimental Binding Affinity of g14-3-3 on Synthetic Phosphopeptides. Binding affinity of the recombinant T214E-g14-3-3 mutant and the WT-g14-3-3 for selected phosphopeptides was calculated based on PepSpot overlay assays data, reported previously.27 Binding affinity of the recombinant protein for each phosphopeptide was expressed as spot signal intensity, measured by densitometric analysis using ImageStudio Lite 5.2 software (LI-COR Biotechnology-GmbH, Germany). Values were expressed as the percentage (%) having the background set as 0% and the spot signal intensity for the reference phosphopeptide Raf1S261, PKINRSApSEPSLHRA, set as 100%. The difference between values obtained for the T214E-g14-3-3 mutant and the WTg14-3-3 (a higher value indicates a stronger binding affinity of the T214E-g14-3-3 mutant) is reported in Table 2. Electrostatic Surface Calculation. The electrostatic surface for g14-3-3 in the “closed” and “open” conformations was calculated with APBS 1.3 with the nonlinear Poisson−

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RESULTS Effect of g14-3-3 Phosphorylation on the Local Conformation of the α8-α9 Loop. To get deeper insight into the relevance of Thr214 phosphorylation on the g14-3-3 conformation, we first investigated its local effect on the α8-α9 loop containing the phosphorylation site. To this aim, SGLD simulations were used, in which a guiding force is introduced to accelerate energy barrier overcoming and to allow achieving fast convergence in conformational sampling.56,57 To also evaluate the possible impact of side chain steric hindrance, three simplified tripeptides corresponding to the g14-3-3 phosphorylation region were used: (i) Gly-Thr-Gly, (ii) Ala-Thr-Ala, and (iii) Leu-Thr-Glu, this latter reproducing the tripeptide sequence centered on Thr214 of WT-g14-3-3. Each model was investigated having threonine in the phosphorylated (pThr) and the nonphosphorylated form. In addition, based on the well-known phosphomimetic properties of glutamic acid, the effect of the substitution of threonine with glutamic acid was also investigated in each model. The frequency of dihedral angles Phi (Φ) and Psi (Ψ) was monitored during 200 ns of SGLD simulation performed in explicit water solvent. By plotting dihedral angles frequency in a Ramachandran-like graph, local conformational preferences emerged, according to chemical properties and steric hindrance of amino acids D


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Journal of Chemical Information and Modeling combinations (Figure 1). Overall, tripeptides phosphorylation induces a remarkable conformational restraint with respect to nonphosphorylated tripeptides. Particularly, in the Gly-Thr-Gly tripeptide, the restriction of dihedral angles conformation was less pronounced than in the Ala-Thr-Ala tripeptide, where side chain introduction coupled with Thr phosphorylation resulted in a remarkable dihedral angles conformational restraint. Indeed, in the Ala-pThr-Ala tripeptide a combination of Φ and Ψ angles (Φ = +60°, Ψ = −25°) was not accessible in SGLD. The g14-3-3 Leu-Thr-Glu tripeptide exemplified the dihedral angles freezing, as in this model only a dihedral angles combination (Φ = −65°, Ψ = −50°) was allowed upon phosphorylation. Interestingly, in all tripeptides a highly comparable conformational restraint was observed upon Thr to Glu mutation. Nevertheless, the mutation of Thr to Glu in the Ala-Thr-Ala tripeptide is not sufficient to fully mimic phosphorylation, whereas it remarkably increases the conformational freezing in the g14-3-3 Leu-Thr-Glu tripeptide, most likely as a consequence of charge repulsion between the side chains of the two adjacent glutamic acid residues. Effect of Phosphorylation on the Global Conformation of g14-3-3. To investigate the effect of Thr214 phosphorylation on the g14-3-3 global conformation, classical MD simulations were performed on dimeric g14-3-3. Starting from the crystallographic structure of the apo g14-3-3 in the dimeric “open” conformation (WT-g14-3-3, PDB ID: 4F7R29), two additional systems were modeled in silico by phosphorylating Thr214 residue (pThr214-g14-3-3) and introducing the phosphomimetic T214E mutation (T214E-g14-3-3) respectively. To increase the statistical significance of our approach, four independent replicas of 200 ns of unrestrained MD simulations in explicit water solvent were performed for each system, starting from slightly different initial coordinates. As attested by the all-atom root-mean-square deviation (RMSD) of Figure 2A, pThr214-g14-3-3 and T214E-g14-3-3 systems reached geometrical convergence after about 50 ns, even if the final RMSD values suggest that a large-scale structural rearrangement has occurred. In contrast, the WT-g14-3-3 system fluctuates around low RMSD values without achieving geometric convergence, thus suggesting that WT-g14-3-3, despite its higher conformational freedom, does not undergo any significant conformational changes in MD simulations. In fact, only Thr214 phosphorylation or T214E mutation proved to impact the global g14-3-3 conformational stability in MD trajectories. Afterward, to identify the regions of g14-3-3 endowed with higher flexibility, per-residue root means square fluctuation (RMSF) of all systems was calculated along MD trajectories (Supporting Information, Figure S1). Residues included in the loops between helices, particularly loops α3-α4, α5-α6, and α8α9, exhibited a higher fluctuation than residues in α-helices. The largest fluctuation was observed for residues 191−237, encompassing the α8-α9 phosphorylatable loop and the α9 helix at the C-terminus of g14-3-3. Notably, the WT-g14-3-3 system exhibited the highest RMSF values, thus confirming its higher conformational freedom when compared to pThr214g14-3-3 and T214E-g14-3-3. To further investigate the conformational stability of the highest fluctuation area corresponding to the α8-α9 phosphorylation loop and the α9 helix (residues 191−237) with respect to the remaining portion of the protein (residues 1−190), RMSD values for each of these regions were separately calculated. As shown in Figure 2(B-D), in all simulated systems RMSD values of amino acids

Figure 2. Analysis of MD trajectories. A) RMSD plotted as a function of MD simulation time. RMSD values are shown for WT-g14-3-3 (black line), pThr214-g14-3-3 (red line), and T214E-g14-3-3 (green line). B, C, D) Comparison between RMSD values of the high fluctuation portion (191−237, black lines) and the rest of the g14-3-3 protein (1−190, red lines) on (B) WT-g14-3-3, (C) pThr214-g14-3-3, and (D) T214E-g14-3-3.

belonging to the α8-α9 loop and the α9 helix are significantly larger than those of the rest of the protein. The remarkable flexibility of the g14-3-3 C-terminal region was also confirmed by B-factors calculation and secondary structure assignment using the dictionary of secondary structure of proteins (DSSP) method (Supporting Information, E


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Journal of Chemical Information and Modeling Figures S2, S3),58 even if this analysis could not account for packaging effects present in the crystal lattice. Despite an overall stability of α-helices in all systems, the C-terminal region clearly exhibited significant structural variations. Analysis of protein backbone evolution during the MD time was also performed (Supporting Information, Figure S4), and comparable results to those of all-atoms calculation were obtained, suggesting that global RMSD fluctuations are largely caused by main-chain structural rearrangement. To further analyze the conformational landscape of whole systems, MD trajectories were submitted to main-chain-based average-linkage clustering, and the reference structures of the most populated cluster were compared to each other. The superposition of pThr-g14-3-3 or the T214E-g14-3-3 representative structure with crystallographic apo g14-3-3 clearly confirmed that the α8-α9 loop and the α9 helix underwent significant conformational change (Figure 3A-B). Particularly, “open-to-closed” conformational

transition occurred in the pThr214-g14-3-3 and T214E-g14-3-3 MD simulations, moving these systems toward the canonical W-like shaped binding conformation of 14-3-3 proteins (Supporting Information, Figure S5). In the case of WT-g14-3-3, representative conformations obtained by each MD replica were significantly different from each other, confirming the unstable nature of this system. However, we did not observe the “open-to-closed” transition in MD replicas of WT-g14-3-3, thus suggesting that large-scale conformational changes may occur in g14-3-3 only upon PTMs or binding to client phosphopeptides. In order to evaluate the time evolution of the “open-to-closed” transition, mass centers distances of α3 and α9 helices along whole pThr214-g14-3-3 and T214E-g14-3-3 MD trajectories was also monitored. As shown in Figure 3C, both systems evolve toward the “closed” conformation, although Thr214 phosphorylation induces a faster “open-to-closed” transition than T214E mutation in MD trajectories. Hydrophobicity-Affinity Relationship of Phosphopeptide-Binding. In previous works, we have shown that the recombinant phosphomimetic T214E mutant has a different affinity than wild type recombinant g14-3-3 toward a subset of synthetic phosphopeptides reproducing known 14-3-3 binding sequences of human and Drosophila proteins.27 To probe whether the change of g14-3-3 affinity may be related to chemical or physicochemical determinants of phosphopeptides, the Engelman scale (also known as GES-scale) was used to quantify their hydrophobicity.59 According to the Engelman scale, a hydrophobicity scoring value was calculated as the sum of individual amino acid hydrophobic contributions and was used herein to analyze phosphopeptides binding to g14-3-3. Particularly, we selected phosphopeptides derived from human kinase suppressor of Ras-1 (KSRS297), Bcl2-associated agonist of cell death (BADS136), M-phase inducer phosphatase 3 (CDC25CS216) wild type as well as eight single- and multiplepoints alanine mutants, and Slowpoke binding protein (SLOBS54), for which the T214E mutant displayed increased binding affinity (Supporting Information, Table S1). Moreover, we selected the phosphopeptide KSRS392, the phosphopeptides derived from Tuberin protein (TSC2S1210), and apoptosis signal-regulating kinase 1 (ASK1S967), for which T214E variant displayed a decreased binding affinity (Supporting Information, Table S1).27 The preliminary analysis performed on these phosphopeptides showed that sequences endowed with a stronger binding affinity to the g14-3-3 T214E mutant were characterized by hydrophobicity scores comprised between −17.9 and −34.1, thus exhibiting overall a balanced hydrophobic/hydrophilic character. In contrast, phosphopeptides exhibiting a weaker binding affinity upon g14-3-3 T214E mutation displayed unbalanced hydrophobic/hydrophilic features. Indeed, the phosphopeptide KSRS392 showed the most polar character (hydrophobicity score = −51.6); while TSC2S1210 was the most hydrophobic among all analyzed phosphopeptides (hydrophobicity score = 9.1), and ASK1 showed a hydrophobicity score significantly higher than highaffinity peptides (hydrophobicity score = −5.9). It is worth mentioning that the phosphorylation independent 14-3-3 binding peptide R18 was included in the analysis, showing a hydrophobicity score of −22 that nicely fits with the confidential limits used herein. Overall, for these phosphopeptides a good agreement between computed hydrophobicity and experimental affinity values was observed (Figure 4, red and green bars).

Figure 3. A-B: g14-3-3 “open-to-closed” transition of (A) pThr214g14-3-3 and (B) T214E-g14-3-3 systems from the initial MD structure (gray) to the representative structure (magenta and green respectively). The conformational transition, which mostly involved the α8-α9 loop and the α9 helix, is highlighted by the black arrow. MD simulations were performed on the dimeric structure of g14-3-3, and the conformational transition was observed in both monomers. For clarity of representation a single monomer is shown in the figure. C) Time evolution of the distance between the mass centers of α3 and α9 helices as a function of MD simulation time. Distances were calculated for pThr214-g14-3-3 and T214E-g14-3-3 systems. F


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Figure 4. Hydrophobicity of selected peptides. In red are highlighted peptides that showed a lower binding affinity for g14-3-3 upon T214E mutation; in green are shown the phosphopeptides having an increased affinity for g14-3-3 upon T214E mutation. In blue are shown the phosphorylated 14-3-3 binding sites present in g14-3-3 interacting proteins having hydrophobicity scoring values within the confidential limits used in this work, as well as the 14-3-3 phosphorylation independent interacting peptide R18 having a hydrophobicity score = −22.

Accordingly, the hydrophobicity scoring value and the confidence limits were furthermore used to understand molecular determinants relevant for binding to g14-3-3 on a panel of phosphopeptides reproducing putative 14-3-3 binding sites present in g14-3-3 interacting proteins experimentally identified in G. duodenalis (Supporting Information, Table S1).60 Indeed, these peptides belong to g14-3-3 client proteins for which the exact g14-3-3 binding site was not determined and were previously highlighted in a bioinformatics study.60 Analysis of hydrophobicity values for these peptides showed that, among the possible g14-3-3 binding sites previously identified, only some of them had a balanced hydrophobic/ hydrophilic character within the above hydrophobicity limits and may be therefore suitable for interacting with the g14-3-3 (Figure 4, blue bars). The only deviation from this numeric rule was observed for the 14-3-3 binding site identified in STE20 kinase (gSTE20S61, Supporting Information, Table S1), which has a hydrophobicity scoring value outside the above-described limits (hydrophobicity score = −11.8). Results of this algebraic operation provide further insight into the understanding of g14-3-3 PPIs and represent a rational approach for the identification and ranking of g14-3-3 binding sequences among the interacting clients. Taken together, these results suggest that hydrophobic/ hydrophilic balance and electrostatic properties of phosphopeptides could play an important role in g14-3-3 recognition. Based on the outcomes of MD simulations described above and showing that the “closed” conformation is suitable for binding to phosphopeptides, we checked whether the “open-to-closed” conformational change might be responsible for alterations in g14-3-3 electrostatic surface potential. As reported in Figure 5, the “closed” conformation displayed the typical electrostatic potential surface of the 14-3-3 amphipathic binding groove, whereas the “open” conformation exposes a considerable hydrophobic patch instead. Therefore, we hypothesize herein that phosphopeptides endowed with balanced hydrophobic/ hydrophilic character may have high electrostatic complementarity with the “closed” conformation of g14-3-3, thus highlighting the key structural role of Thr214 phosphorylation

Figure 5. (A) Distribution of the electrostatic potential on the solvent surface accessible area of the pThr214-g14-3-3 system (“closed” conformation) and (B) the crystallographic apo structure (PDB ID: 4F7R) of g14-3-3 (“open” conformation). The molecular surface is colored according to electrostatic potential from positive (blue) to negative (red).

as well as T214E phosphomimetic mutation in binding to phosphopeptides. X-ray Crystal Structure of Phosphopeptide-Bound g14-3-3. Although g14-3-3/clients interaction was found to be crucial for the G. duodenalis life cycle, its structural details have not yet been elucidated. Since the knowledge of g14-3-3 phosphopeptide-bound conformation is essential to correctly identify key residues involved in PPIs and to guide further ligand design approaches, the X-ray crystallographic structure of g14-3-3 bound to synthetic phosphopeptide was solved. To this aim, we used the phosphorylated mode-1 octapeptide A8Ap28 and the recombinant g14-3-3-polyG10, a g14-3-3 mutant in which the last two C-terminal residues were replaced by a stretch of 10 glycines thus partially mimicking the posttranslational C-terminal polyglycylation.29 Since expressed in bacteria, this recombinant protein, as well as the WT-g14-3-3, was not phosphorylated at Thr214 (see the Supporting Information and Figure S6). Indeed, despite several attempts, we were not able to obtain diffraction quality crystals of the recombinant WT-g14-3-3 in complex with A8Ap, whereas the crystallographic structure of g14-3-3-polyG10 in the apo form and in complex with A8Ap was obtained. Apo g14-3-3-polyG10 (PDB ID: 5BY9) was solved at low resolution (4 Å), crystallized in different conditions but with G


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correct positioning of the phosphopeptide within the amphipathic groove. The absence of domain swapping and the inability to form fibers in the g14-3-3-polyG10/A8Ap crystal are likely the result of the combined effect of (i) the C-terminal polyglycine mimic, hampering the exchange of the α9 helix between monomers, with (ii) phosphopeptide binding, which favors the folding of the α9 helix toward the binding cleft by involving contacts between the phosphopeptide backbone and residues in the helix (i.e., Asn231). To monitor the stability of the g14-3-3-polyG10/A8Ap complex, two independent replicas of 200 ns MD simulations were performed in explicit water solvent. RMSD calculation on backbone atoms revealed that the system reached geometric convergence quickly (Supporting Information, Figure S10), thus denoting the higher conformational stability of the g14-33-polyG10/A8Ap complex with respect to g14-3-3 in the apo form. The consistency of these theoretical results was further evaluated by comparing residues fluctuation (RMSF) in the crystal structure and in MD trajectories (Supporting Information, Figure S11). The representative structure of MD trajectories (see Materials and Methods) was compared with the “closed” conformation obtained from MD simulations described above, showing that all systems are endowed with a largely superimposable conformation (backbone RMSD < 1.5 Å) (Figure 6). Notably, the highest variation between these

the same symmetry and cell constants of the apo g14-3-3 previously solved (PDB ID: 4F7R).29 The two structures are almost identical (RMSD 0.62 Å), although in both cases the last ten C-terminal residues, plus the polyglycine stretch, could not be modeled. Both proteins crystallize as classical dimer, with dimerization mediated by interaction between N-termini, but with the C-termini in the “open” conformation (Supporting Information, Figure S7). This “open” conformation, as already proved for the apo WT-g14-3-3, allows α9 to be exchanged (swapped) between adjacent dimers, leading to a further Cterminal dimerization, oligomerization, and fibrils formation in the crystal as well as in solution. C-terminal mediated oligomerization does not take place for the endogenous g143-3 (present in G. duodenalis and constitutively phosphorylated) since it is prevented by post-translational polyglycylation of the C-terminus.25,27,29 Indeed, polyglycylation mimic alone (as occur for the g14-3-3-polyG10 mutant) can partially prevent oligomerization of recombinant g14-3-3.29 The structure of g14-3-3-polyG10 in complex with the phosphopeptide A8Ap, solved at 3.1 Å resolution, comprises two functional dimers per asymmetric unit, where each monomer possesses a client-binding groove accommodating a phosphopeptide in extended conformation. The superimposition of the backbone Cα atoms of the four monomers yields a RMSD ranging from 0.66 to 0.73 Å, indicating a nearly identical 3D arrangement. The main differences concern the α8-α9 loop, also displaying a higher B-factor and a less defined electron density than the rest of the structure. The polyglycine stretch was not visible in any of the monomers, probably as a consequence of its intrinsic mobility. Both dimers are folded into the typical W-like shape: the classical N-terminal dimerization occurs through the same interactions already described for the apo structure.29 More important, each monomer of the dimeric unit displayed the α9 helix in the “closed” conformation. Accordingly, the C-terminal dimerization mediated by the α9 helix swapping to the “open” conformation,29 as observed in both apo g14-3-3 crystal structures, was not observed herein (Supporting Information, Figure S8). The binding mode of the phosphopeptide A8Ap is the same in all monomers (Supporting Information, Figures S8, S9) and resembles that observed in other 14-3-3/phosphopeptide solved complexes, confirming our previous predictions based on the comparison of the structures of the apo-g14-3-3 and h14-3-3ε-peptide complex (pdb: 2BR9).29 It should be noted that only four out of eight residues of A8Ap (ApSAP) could be modeled in the electron density map, indicating that the other residues have great mobility and poorly participate in the interaction. The major contribution to phosphopeptide binding is due to the positive surface region comprising highly conserved residues Arg60, Arg135, and Tyr136 (namely Arg57, Arg130, and Tyr131 in PDB: 2BR9 numbering), which directly establish an extensive H-bonding network with the phosphoserine of the peptide (Supporting Information, Figures S8, S9). Consistent with the known binding mode of targets to 14-3-3s, H-bond interactions are also established between the side chain of Asn180 and Asn231 (Asn176 and Asn227 in PDB: 2BR9) and the phosphopeptide main-chain atoms of residues Ala +1 and Ala −1, respectively (numbering is related to phosphoserine).61,62 Other positive residues such as Lys53 and Arg64 (Lys50 and Arg64 in PDB: 2BR9) participate in the hydrogen bonding network established upon phosphopeptide binding and are most likely involved in the

Figure 6. (A) Superimposition of g14-3-3 structures relaxed by MD simulations: g14-3-3-polyG10/A8Ap (magenta), pThr214-Apo (cyan), and T214E-Apo (green). (B) Helix α9 conformational arrangements are highlighted (the same colors as in (A) are used to show different g14-3-3 structures).

structures was found in the α8-α9 loop, which exhibits a high degree of conformational flexibility. Moreover, this loop has been also proposed as a key region for target specificity in human 14-3-3σ.63 Theoretical Binding Affinity of g14-3-3 Synthetic Phosphopeptides. To check whether differences in the hydrophobic/hydrophilic character of phosphopeptides may correspond to different delta energy of binding to g14-3-3, the newly solved g14-3-3-polyG10/A8Ap structure was phosphorylated in silico at Thr214, or Thr214 was mutated to Glu, while A8Ap was used as a template to model the 15-mer phosphopeptides KSR S297 , CDC25C S216 , KSR S392 , and TSC2S1210. These peptides were selected because they showed a significant variation of affinity upon phosphomimetic T214E mutation in previous experiments.27 Two independent replicas of 200 ns MD simulations of each g14-3-3/phosphopeptide system were performed in explicit water solvent. RMSD analysis showed that all complexes are stable and reached geometric convergence after just a few nanoseconds of unrestrained MD (Supporting Information, Figure S12). Representative structures of MD trajectories were subsequently H


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promote the stabilization of the “closed” conformation of g143-3. Interestingly, conformational freezing is clearly shown also in the Thr to Glu mutants, confirming the phosphomimetic nature of the glutamate side chain. MD results showed that Thr214 phosphorylation or T214E mutation induces a conformational rearrangement that leads to a “closed” and stable g14-3-3 conformation, which highly resembles the canonical phosphopeptide binding conformation of 14-3-3s.6 In contrast, MD studies highlighted the high flexibility of apo WT-g14-3-3, which never achieves geometric convergence and does not undergo large-scale conformational changes. This different behavior further highlights the relevance of PTMs at Thr214 in favoring the “open-to-closed” transition of g14-3-3. Furthermore, even if glutamate replacement to pThr214 does not completely mimic phosphorylation in the full-length g14-33, our findings support the in vitro experimental evidence and the significance of T214E mutation as a valuable tool to investigate g14-3-3 peptides binding properties.15 In accordance with previous experimental observations,44 here we were able to link the effects of Thr214 phosphorylation on phosphopeptides binding affinity with the chemical and physicochemical properties of phosphopeptides. The comparison of Engelman hydrophobicity values calculated for each phosphopeptide, including those present in the g14-3-3 target proteins from G. duodenalis and the reference 14-3-3 binding peptide R18, clearly shows that hydrophobic/hydrophilic features play a key role in g14-3-3 binding affinity. In particular, phosphopeptides endowed with a balanced character have a greater affinity for the “closed” conformation of g14-3-3 induced upon Thr214 phosphorylation than phosphopeptides with unbalanced features. According to our MD simulation and electrostatic surface calculation, this was because of the higher electrostatic complementarity of phosphopeptides with the g14-3-3 amphipathic binding site. Therefore, g14-3-3 phosphorylation at Thr214, or the phosphomimetic T214E mutation, proved to be crucial for promoting the conformational transition from the apo “open” to the “closed” peptidebound form of g14-3-3, which exposes an electrostatic surface potential suitable for phosphopeptides binding. Here, we also present the X-ray crystallographic structure of the g14-3-3-polyG10, a mutant mimicking the C-terminal g143-3 polyglycine chain,17 both in the apo form and in complex with the phosphorylated octapeptide A8Ap. As for the apo WTg14-3-3, the g14-3-3-polyG10 crystallized in an oligomeric conformation, as a consequence of the α9 helix swapping. Although the addition of ten glycines to the C-terminus has been shown to favor the dimeric vs oligomeric state of the protein, the crystallization process seems to shift the equilibrium in the opposite direction, i.e. toward the oligomeric form. Although we have established that 14-3-3 binding to phosphopeptide may potentially occur with the “classical” dimer as well as the open oligomer,29 the dimer/oligomer ratio of unmodified recombinant g14-3-3 is not affected in solution by peptide binding. Noteworthy, in our crystallization conditions, the combination of the reduced oligomerization tendency of g14-3-3-polyG10 in association with phosphopeptide binding can easily explain why the g14-3-3-polyG10 complex with the peptide crystallized in the dimeric “closed” conformation but not the WT-g14-3-3. Although g14-3-3 can bind to phosphopeptide both in the presence and in the absence of Thr214 phosphorylation,29 our crystallographic results proved that the g14-3-3 binds to phosphopeptide in the “closed” conformation and not in the swapped one (i.e., “open”

subjected to visual inspection. Overall, higher affinity phosphopeptides KSRS297 and CDC25CS216 establish a larger number of H-bonds with g14-3-3 residues compared to KSRS392 and TSC2S1210, in particular Arg45, Asn46, Ser49, Lys128, and Asn231. Interestingly, CDC25CS216 is the only peptide able to form a bidentate H-bond between Arg at position +7 (numbering refers to the pSer position) and pThr214 (Supporting Information, Figure S13). The delta energy of binding (ΔG) of these phosphopeptides to pThr214-g14-3-3 and T214E-g14-3-3 was calculated along the MD trajectory by means of the Molecular Mechanics Poisson−Boltzmann Surface Area (MM-PBSA) method.64 As summarized in Table 2, a good agreement between computational and in Table 2. Binding Free Energies Calculated for Selected Peptidesb

peptide KSRS297 CDC25CS216 KSRS392 TSC2S1210

ΔG (MM-PBSA) kcal/mol (±SEM) pThr214-g14-3-3 −40.22 −58.67 −17.78 −28.63

± ± ± ±

1.39 2.32 2.16 1.68

ΔG (MM-PBSA) kcal/mol (±SEM) T214E-g14-3-3 −39.60 −66.73 −13.49 −28.64

± ± ± ±

2.20 1.86 2.53 3.62

binding affinity (%) (T214E-g143-3 − WT-g14-3-3)a 36.6 22.1 −10.7 6.2

± ± ± ±

0.5 0.7 0.7 0.3

a In vitro phosphopeptide-binding affinity difference of T214E-g14-3-3 mutant minus WT-g14-3-3. Values were calculated and expressed as described in the Materials and Methods. bSEM = standard error of the mean.

vitro data was observed, as the theoretical and experimental affinity of KSRS297 and CDC25CS216 is significantly higher than that of KSRS392 and TSC21210. Notably, CDC25CS216 was able to interact by H-bond with pThr214 in MD simulations, thus suggesting that this interaction could be a unique feature in the rational design of high-affinity small-molecules or peptides that selectively interact with g14-3-3. Energy decomposition further shed light on the forces that drive the interaction between these peptides and pThr214-g14-3-3, in agreement with previous findings (Supporting Information, Table S2). Finally, also in this case the consistency of MD results was further evaluated by comparing residues fluctuation (RMSF) in the newly solved crystal structure and in MD trajectories (Supporting Information, Figure S11).

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DISCUSSION In the present study, we deeply investigated the implication of Thr214 phosphorylation on the g14-3-3 protein. We first explored the local consequences of Thr214 phosphorylation by performing SGLD simulations on simplified tripeptides that are representative of the Thr214 phosphorylation region. In our previous work,29 we have hypothesized that restriction of the conformational freedom of the α8-α9 loop upon Thr214 phosphorylation may be promoted by the interaction of the phosphate moiety with some g14-3-3 residues, thus favoring a more stable fold of helix α9 toward the binding groove as well as the acquisition of a “closed” conformation of g14-3-3. Results of the present study on tripeptide models unequivocally suggest that Thr214 phosphorylation may have remarkable effects on the α8-α9 loop flexibility, thus resulting in a dihedral angles restraint that is proportional to side chain steric hindrance without necessarily requiring the interaction with flanking g143-3 residues. Of course, intramolecular H-bonds may further stabilize the restricted conformation of the α8-α9 loop and I



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with the exchange of helices α9 between distinct g14-3-3 dimeric units). Indeed, the conformation adopted by g14-3-3polyG10 in complex with the A8Ap phosphopeptide supports the “open-to-closed” conformational rearrangement shown by MD studies that occurs after phosphorylation of Thr214 and thus promoting target binding (Figure S14). Furthermore, the solved structure provides information on the phosphopeptide binding properties of g14-3-3, giving direct evidence that the key residues Lys53, Arg60, Arg135, and Tyr136 are involved in g14-3-3 binding to the phosphorylated residue, as already observed in other 14-3-3 target complexes.6 The structure-based modeling of the binding conformation of phosphopeptides displaying either high-affinity (KSRS297 and CDC25CS216) and low-affinity (KSRS392 and TSC2S1210) for g14-3-3, together with the extended MD simulations, highlighted how the theoretical affinity nicely correlates with previous experimental observations.44 In addition to Lys53, Arg60, Arg135, and Tyr136, our approach indicates that other residues in the amphipathic binding groove, such as Arg45, Asn46, Ser49, Lys128, and Asn231, form H-bonds with the phosphopeptides and can be key pharmacophoric hot-spots of high-affinity peptides. In contrast, conformational instability and unbalanced hydrophobic/hydrophilic features are associated with low-affinity peptides. Intriguingly, the establishment of a H-bond between the phosphorylated Thr214 and a residue with a positive net charge in the target binding site, such as the arginine at position +7 in CDC25CS216, could represent a specific feature relevant to the design of small molecules selectively acting on g14-3-3 PPIs. This H-bond may resemble the effect of a divalent cation, such as Mg2+ or polyamines, in promoting the interaction between the plant 14-3-3 isoform GF14ω and phosphorylated nitrate reductase by binding to the α8-α9 loop.65 Indeed, although the main interface allowing ligand binding is mediated by the highly conserved amphipathic groove of 14-3-3s, structural data from previous studies indicate that isoform(s) specific interactions are strongly affected by the 14-3-3 protein regions, including the surface formed by helices α8 and α9, which presumably dictate ligand and dimerization preferences.63,66−68 Similarly, a molecular tweezer has been shown to target Lys 214 of human 14-3-3σ, located at the beginning of the helix α9 and facing the central binding channel, resulting in an inhibitory effect on 143-3 binding to two clients.69 Taken together, our results provide a further and structural understanding of g14-3-3 features and set the basis of subsequent drug design studies. Indeed, the design of small molecules interfering with g14-3-3 is highly desirable in antigiardial drug discovery. Pharmacophoric, electrostatic, and energetic features here described for phosphopeptides, such as the suitable hydrophobic/hydrophilic character or the interaction with phosphorylated Thr214, may be easily exploited in the design of small molecules inhibitors of g14-3-3 PPIs. Moreover, outcomes of the crystallographic study and the MD simulations underscore the relevance of PTMs in the g14-3-3 structure and provide key elements for approaching the structure-guided design of targeting modulators. These molecules may be valuable tools in further understanding the functions of g14-3-3 as well as valuable hit or lead compounds for further optimization as therapeutic agents.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.5b00452. Additional details on MD simulation, including RMSF and RMSD graphs, B-factor calculation, DSSP-calculated secondary structure changes, superimposition of representative structures; crystallographic data, mass spectra analysis of recombinant g14-3-3, and details of the phosphopeptides panel (PDF) Movie describing the Minimum Action path from crystallographic apo to crystallographic peptide-bound g14-3-3 (MPG) Movie describing the Minimum Action path from crystallographic apo to the final frame of MD trajectory of g14-3-3 phosphorylated at Thr214 (MPG) Movie describing “open-to-closed” transition path of g14-3-3 fully simulated by MD (MPG) Movie describing all the paths compared to each other (MPG) Accession Codes

Atomic coordinates have been deposited in the Protein Data Bank (PDB) (www.rcsb.org/pdb/home/home.do). PDB ID Codes: 4ZQ0, 5BY9.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: 39 0577 234306. Fax: 39 0577 234333. E-mail: botta. [email protected] (M.B.). *Phone: 39 06 4990 2670. Fax: 039 06 4990 3561. E-mail: [email protected] (M.L.). Author Contributions ∇

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Dr. Serena Camerini, Istituto Suoperiore di Sanità, Rome, Italy, for the priceless help in mass spectroscopy analysis. The authors gratefully acknowledge European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N 283570) and Bag Project 3959. The authors thank HZB (Helmholtz Zentrum Berlin) for allocation of synchrotron radiation beam-time. This study has been partially supported by Istituto Superiore di Sanità (project 14A1/678/2014).

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REFERENCES

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