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The prediction of protein side chain conformations from backbone coordinates is an important task in structural biology, with applications in structure prediction and protein design. It is a difficult problem due to its combinatorial nature. We study the performance of an "MMGBSA" energy function, implemented in our protein design program Proteus, which combines molecular mechanics terms, a Generalized Born and Surface Area (GBSA) solvent model, with approximations that make the model pairwise additive. Proteus is not a competitor to specialized side chain prediction programs due to its cost, but it allows protein design applications, where side chain prediction is an important step and MMGBSA an effective energy model. We predict the side chain conformations for 18 proteins. The side chains are first predicted individually, with the rest of the protein in its crystallographic conformation. Next, all side chains are predicted together. The contributions of individual energy terms are evaluated and various parameterizations are compared. We find that the GB and SA terms, with an appropriate choice of the dielectric constant and surface energy coefficients, are beneficial for single side chain predictions. For the prediction of all side chains, however, errors due to the pairwise additive approximation overcome the improvement brought by these terms. We also show the crucial contribution of side chain minimization to alleviate the rigid rotamer approximation. Even without GB and SA terms, we obtain accuracies comparable to SCWRL4, a specialized side chain prediction program. In particular, we obtain a better RMSD than SCWRL4 for core residues (at a higher cost), despite our simpler rotamer library. Proteins 2016; 84:803-819. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled., (© 2015 Wiley Periodicals, Inc.)

The GTPase aIF5B is a universally conserved initiation factor that assists ribosome assembly. Crystal structures of its nucleotide complexes, X-ray(GTP) and X-ray(GDP), are similar in the nucleotide vicinity, but differ in the orientation of a distant domain IV. This has led to two, contradictory, mechanistic models. One postulates that X-ray(GTP) and X-ray(GDP) are, respectively, the active, "ON" and the inactive, "OFF" states; the other postulates that both structures are OFF, whereas the ON state is still uncharacterized. We study GTP/GDP binding using molecular dynamics and a continuum electrostatic free energy method. We predict that X-ray(GTP) has a ≈ 3 kcal/mol preference to bind GDP, apparently contradicting its assignment as ON. However, the preference arises mainly from a single, nearby residue from the switch 2 motif: Glu81, which becomes protonated upon GTP binding, with a free energy cost of about 4 kcal/mol. We then propose a different model, where Glu81 protonation/deprotonation defines the ON/OFF states. With this model, the X-ray(GTP):GTP complex, with its protonated Glu81, is ON, whereas X-ray(GTP):GDP is OFF. The model postulates that distant conformational changes such as domain IV rotation are "uncoupled" from GTP/GDP exchange and do not affect the relative GTP/GDP binding affinities. We analyze the model using a general thermodynamic framework for GTPases. It yields rather precise predictions for the nucleotide specificities of each state, and the state specificities of each nucleotide, which are roughly comparable to the homologues IF2 and aIF2, despite the lack of any conformational switching in the model., (Copyright © 2012 Wiley Periodicals, Inc.)

Archaeal Initiation Factor 2 is a GTPase involved in protein biosynthesis. In its GTP-bound, "ON" conformation, it binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and their dependence on the conformational state, molecular dynamics free energy simulations were performed. The ON state specificity was predicted to be weak, with a GTP/GDP binding free energy difference of -1 kcal/mol, favoring GTP. The OFF state specificity is larger, 4 kcal/mol, favoring GDP. The overall effects result from a competition among many interactions in several complexes. To interpret them, we use a simpler, dielectric continuum model. Several effects are robust with respect to the model details. Both nucleotides have a net negative charge, so that removing them from solvent into the binding pocket carries a desolvation penalty, which is large for the ON state, and strongly disfavors GTP binding compared to GDP. Short-range interactions between the additional GTP phosphate group and ionized sidechains in the binding pocket offset most, but not all of the desolvation penalty; more distant groups also contribute significantly, and the switch 1 loop only slightly. The desolvation penalty is lower for the more open, wetter OFF state, and the GTP/GDP difference much smaller. Short-range interactions in the binding pocket and with more distant groups again make a significant contribution. Overall, the simulations help explain how conformational selection is achieved with a single phosphate group., (Copyright © 2012 Wiley Periodicals, Inc.)

Computational Protein Design (CPD) is a promising method for high throughput protein and ligand mutagenesis. Recently, we developed a CPD method that used a polar-hydrogen energy function for protein interactions and a Coulomb/Accessible Surface Area (CASA) model for solvent effects. We applied this method to engineer aspartyl-adenylate (AspAMP) specificity into Asparaginyl-tRNA synthetase (AsnRS), whose substrate is asparaginyl-adenylate (AsnAMP). Here, we implement a more accurate function, with an all-atom energy for protein interactions and a residue-pairwise generalized Born model for solvent effects. As a first test, we compute aminoacid affinities for several point mutants of Aspartyl-tRNA synthetase (AspRS) and Tyrosyl-tRNA synthetase and stability changes for three helical peptides and compare with experiment. As a second test, we readdress the problem of AsnRS aminoacid engineering. We compare three design criteria, which optimize the folding free-energy, the absolute AspAMP affinity, and the relative (AspAMP-AsnAMP) affinity. The sequences and conformations are improved with respect to our previous, polar-hydrogen/CASA study: For several designed complexes, the AspAMP carboxylate forms three interactions with a conserved arginine and a designed lysine, as in the active site of the AspRS:AspAMP complex. The conformations and interactions are well maintained in molecular dynamics simulations and the sequences have an inverted specificity, favoring AspAMP over AsnAMP. The method is not fully successful, since experimental measurements with the seven most promising sequences show that they do not catalyze at a detectable level the adenylation of Asp (or Asn) with ATP. This may be due to weak AspAMP binding and/or disruption of transition-state stabilization., (Copyright © 2011 Wiley-Liss, Inc.)

Computationally designed protein sequences have been proposed as a basis to perform fold recognition and homology searching. To investigate this possibility, an automated procedure is used to completely redesign 24 SH3 proteins and 22 SH2 proteins. We use the experimental backbone coordinates as fixed templates in the folded state and a molecular mechanics model to compute the pairwise interaction energies between all sidechain types and conformations. Energy calculations are done with the Proteins@Home volunteer computing platform. A heuristic algorithm is then used to scan the sequence and conformational space for optimal solutions. We produced 200,000-450,000 sequences for each backbone template. The designed sequences ressemble moderately-distant, natural homologues of the initial templates, according to their identity scores and their similarity with respect to the Pfam sets of SH2 and SH3 domains. Standard homology detection tools document their native-like character: the Conserved Domain Database recognizes 61% (52%) of our low-energy sequences as SH3 (SH2) domains; the SUPERFAMILY, Hidden-Markov Model library recognizes 81% (84%). Conversely, position specific scoring matrices (PSSMs) derived from our designed sequences can be used to detect natural homologues in sequence databases. Within SwissProt, a set of natural SH3 PSSMs detects 772 SH3 domains, for example; our designed PSSMs detect 67% of these, plus one additional sequence and two false positives. If six amino acids involved in substrate binding (a selective pressure not accounted for in our design) are reset to their experimental types, then 77% of the experimental SH3 domains are detected. Results for the SH2 domains are similar. Several directions to improve the method further are discussed.

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand., (2007 Wiley-Liss, Inc.)

Structure prediction and computational protein design should benefit from accurate solvent models. We have applied implicit solvent models to two problems that are central to this area. First, we performed sidechain placement for 29 proteins, using a solvent model that combines a screened Coulomb term with an Accessible Surface Area term (CASA model). With optimized parameters, the prediction quality is comparable with earlier work that omitted electrostatics and solvation altogether. Second, we computed the stability changes associated with point mutations involving ionized sidechains. For over 1000 mutations, including many fully or partly buried positions, we compared CASA and two generalized Born models (GB) with a more accurate model, which solves the Poisson equation of continuum electrostatics numerically. CASA predicts the correct sign and order of magnitude of the stability change for 81% of the mutations, compared to 97% with the best GB. We also considered 140 mutations for which experimental data are available. Comparing to experiment requires additional assumptions about the unfolded protein structure, protein relaxation in response to the mutations, and contributions from the hydrophobic effect. With a simple, commonly-used unfolded state model, the mean unsigned error is 2.1 kcal/mol with both CASA and the best GB. Overall, the electrostatic model is not important for sidechain placement; CASA and GB are equivalent for surface mutations, while GB is far superior for fully or partly buried positions. Thus, for problems like protein design that involve all these aspects, the most recent GB models represent an important step forward. Along with the recent discovery of efficient, pairwise implementations of GB, this will open new possibilities for the computational engineering of proteins., (2007 Wiley-Liss, Inc.)

In zinc proteins, the Zn2+ cation frequently binds with a tetrahedral coordination to cysteine and histidine side chains, for example, in many DNA-binding proteins, where it plays primarily a structural role. We examine the possibility of thiolate protonation in Cys(x)His(y)-Zn2+ groups, both in proteins and in solution, through a combination of theoretical calculations and database analysis. Seventy-five percent of the thiolate-coordinated zincs in the Cambridge Structural Database are tetrahedral, while di-alkanethiol coordination always involves five or more ligands. Ab initio quantum calculations are performed on (ethanethiol/thiolate)(3)imidazole-Zn2+ complexes in vacuum, yielding geometries and gas phase basicities. Protonating one (respectively two) thiolates increases the Zn-S(thiol) distance by 0.4 A (respectively 0.3 A), providing a structural marker for protonation. The stabilities of the complexes in solution are compared by combining the gas phase basicities with continuum dielectric solvation calculations. In a continuum solvent with permittivity epsilon = 4, 20, or 80, one of three thiolates is predicted to be protonated at neutral pH. By extension, Cys4-Zn2+ groups are expected to be protonated in the same conditions. In contrast, most Cys3His and Cys4 geometries in the Protein Data Bank (PDB) appear consistent with all-thiolate Zn2+ coordination. This apparent discrepancy is resolved by two recent surveys of zinc protein structures, which suggest that these all-thiolate sites are stabilized by charged and polar groups nearby in the protein, thus overcoming their intrinsic instability. However, the experimental resolution is not sufficient in all the PDB structures to rule out a thiol/thiolate mixture, and protonated thiolates may occur in some proteins not solved at high resolution or not represented in the PDB, as suggested by recent mass spectrometry experiments; this possibility should be allowed for in X-ray structure refinement., (Copyright 2002 Wiley-Liss, Inc.)

Implicit solvent models are increasingly important for the study of proteins in aqueous solution. Here, the generalized Born (GB) solvent polarization model as implemented in the analytical ACE potential [Schaefer and Karplus (1996) J Phys Chem 100:1578] is used to perform molecular dynamics simulations of two small, homologous proteins: the immunoglobulin-binding domain of streptococcal protein G and the Ras binding domain of Raf. Several model parameterizations are compared through more than 60 ns of simulation. Results are compared with two simpler solvent models-an accessible surface area model and a distant-dependent dielectric model, with finite-difference Poisson calculations, with existing explicit solvent simulations, and with experimental data. The simpler models yield stable but distorted structures. The best GB/ACE implementation uses a set of atomic Voronoi volumes reported recently, obtained by averaging over a large database of crystallographic protein structures. A 20% reduction is applied to the volumes, compensating in an average sense for an excessive de-screening of individual charges inherent in the ACE self-energy and for an undersolvation of dipolar groups inherent in the GB screening function. This GB/ACE parameterization yields stable trajectories on the 0.5-1-ns time scale that deviate moderately (approximately 1.5-2.5 A) from the X-ray structure, reproduce approximately the surface distribution of charged, polar, and hydrophobic groups, and reproduce accurately backbone flexibility as measured by amide NMR-order parameters. Over longer time scales (1.5-3 ns), some of the protein G runs escape from the native energy basin and deviate strongly (3 A) from the native structure. The conformations sampled during the transition out of the native energy basin are overstabilized by the GB/ACE solvation model, as compared with a numerical treatment of the full dielectric continuum model., (Copyright 2001 Wiley-Liss, Inc.)

The protein Raf is an immediate downstream target of Ras in the MAP kinase signalling pathway. The complex of Ras with the Ras-binding domain (RBD) of Raf has been modelled by homology to the (E30D,K31E)-Rap1A:RBD complex, and both have been subjected to multiple molecular dynamics simulations in solution. While both complexes are stable, several rearrangements occur in the Ras:RBD simulations: the RBD loop 100-109 moves closer to Ras, Arg73 in the RBD moves towards Ras to form a salt bridge with Ras-Asp33, and Loop 4 of the Ras switch II region shifts upwards toward the RBD. The Ras:RBD interactions (including the RBD-Arg73 interaction) are consistent with available NMR and mutagenesis data on the Ras: RBD complex in solution. The Ras switch II region does not interact directly with the RBD, although indirect interactions exist through the effector domain and bridging water molecules. No large-scale RBD motion is seen in the Ras:RBD complex, compared to the Rap:RBD complex, to suggest an allosteric activation of Raf by Ras. This may be because the Raf kinase domain (whose structure is unknown) is not included in the model.

Histidyl-tRNA synthetase (HisRS) differs from other class II aminoacyl-tRNA synthetases (aaRS) in that it harbors an arginine at a position where the others bind a catalytic Mg2+ ion. In computer experiments, four mutants of HisRS from Escherichia coli were engineered by removing the arginine and introducing a Mg2+ ion and residues from seryl-tRNA synthetase (SerRS) that are involved in Mg2+ binding. The mutants recreate an active site carboxylate pair conserved in other class II aaRSs, in two possible orders: Glu-Asp or Asp-Glu, replacing Glu-Thr in native HisRS. The mutants were simulated by molecular dynamics in complex with histidyl-adenylate. As controls, the native HisRS was simulated in complexes with histidine, histidyl-adenylate, and histidinol. The native structures sampled were in good agreement with experimental structures and biochemical data. The two mutants with the Glu-Asp sequence showed significant differences in active site structure and Mg2+ coordination from SerRS. The others were more similar to SerRS, and one of them was analyzed further through simulations in complex with histidine, and His+ATP. The latter complex sampled two Mg2+ positions, depending on the conformation of a loop anchoring the second carboxylate. The lowest energy conformation led to an active site geometry very similar to SerRS, with the principal Mg2+ bridging the alpha- and beta-phosphates, the first carboxylate (Asp) coordinating the ion through a water molecule, and the second (Glu) coordinating it directly. This mutant is expected to be catalytically active and suggests a basis for the previously unexplained conservation of the active site Asp-Glu pair in class II aaRSs other than HisRS.

Recognition of Ras by its downstream target Raf is mediated by a Ras-recognition region in the Ras-binding domain (RBD) of Raf. Residues 78-89 in this region occupy two different conformations in the ensemble of NMR solution structures of the RBD: a fully alpha-helical one, and one where 87-90 form a type IV beta-turn. Molecular dynamics simulations of the RBD in solution were performed to explore the stability of these and other possible conformations of both the wild-type RBD and the R89K mutant, which does not bind Ras. The simulations sample a fully helical conformation for residues 78-89 similar to the NMR helical structures, a conformation where 85-89 form a 3(10)-helical turn, and a conformation where 87-90 form a type I beta-turn, whose free energies are all within 0.3 kcal/mol of each other. NOE patterns and H(alpha) chemical shifts from the simulations are in reasonable agreement with experiment. The NMR turn structure is calculated to be 3 kcal/mol higher than the three above conformations. In a simulation with the same implicit solvent model used in the NMR structure generation, the turn conformation relaxes into the fully helical conformation, illustrating possible structural artifacts introduced by the implicit solvent model. With the Raf R89K mutant, simulations sample a fully helical and a turn conformation, the turn being 0.9 kcal/mol more stable. Thus, the mutation affects the population of RBD conformations, and this is expected to affect Ras binding. For example, if the fully helical conformation of residues 78-89 is required for binding, its free energy increase in R89K will increase the binding free energy by about 0.6 kcal/mol.