The Curious Case of A31P, a Topology-Switching Mutant of the Repressor of Primer Protein: A Molecular Dynamics Study of Its Folding and Misfolding.

The effect of mutations on protein structures is usually rather localized and minor. Finding a mutation that can single-handedly change the fold and/or topology of a protein structure is a rare exception. The A31P mutant of the homodimeric Repressor of primer (Rop) protein is one such exception: This single mutation ─and as demonstrated by two independent crystal structure determinations─ can convert the canonical (left-handed/all-antiparallel) 4-α-helical bundle of Rop to a new form (right-handed/mixed parallel and antiparallel bundle) displaying a previously unobserved "bisecting U" topology. The main problem with understanding the dramatic effect of this mutation on the folding of Rop is to understand its very existence: Most computational methods appear to agree that the mutation should have had no appreciable effect, with the majority of energy minimization methods and protein structure prediction protocols indicating that this mutation is fully consistent with the native Rop structure, requiring only a local and minor change at the mutation site. Here we use two long (10 µs each) molecular dynamics simulations to compare the stability and dynamics of the native Rop versus a hypothetical structure that is identical with the native Rop but is carrying this single Alanine31 to Proline mutation. Comparative analysis of the two trajectories convincingly shows that, in contrast to the indications from energy minimization ─but in agreement with the experimental data─, this hypothetical native-like A31P structure is unstable, with its turn regions almost completely unfolding, even under the relatively mild 320 K NpT simulations that we have used for this study. We discuss the implication of these findings for the folding of the A31P mutant, especially with respect to the proposed model of a double-funneled energy landscape.

Folding molecular dynamics simulation of T-peptide, a HIV viral entry inhibitor: Structure, dynamics, and comparison with the experimental data.

Peptide T is a synthetic octapeptide fragment, which corresponds to the region 185-192 of the gp120 HIV coat protein and functions as a viral entry inhibitor. In this work, a folding molecular dynamics simulation of peptide T in a membrane-mimicking (DMSO) solution was performed with the aim of characterizing the peptide's structural and dynamical properties. We show that peptide T is highly flexible and dynamic. The main structural characteristics observed were rapidly interconverting short helical stretches and turns, with a notable preference for the formation of ß-turns. The simulation also indicated that the C-terminal part appears to be more stable than the rest of the peptide, with the most preferred conformation for residues 5-8 being a ß-turn. In order to validate the accuracy of the simulations, we compared our results with the experimental NMR data obtained for the T-peptide in the same solvent. In agreement with the simulation, the NMR data indicated the presence of a preferred structure in solution that was consistent with a ß-turn comprising the four C-terminal residues. An additional comparison between the experimental and simulation-derived chemical shifts also showed a reasonable agreement between experiment and simulation, further validating the simulation-derived structural characterization of the T-peptide. We conclude that peptide folding simulations produce physically relevant results even when performed with organic solvents that were not part of the force field parameterization procedure.

A proof-of-concept study of the secondary structure of influenza A, B M2 and MERS- and SARS-CoV E transmembrane peptides using folding molecular dynamics simulations in a membrane mimetic solvent.

Here, we have carried out a proof-of-concept molecular dynamics (MD) simulation with adaptive tempering in a membrane mimetic environment to study the folding of single-pass membrane peptides. We tested the influenza A M2 viroporin, influenza B M2 viroporin, and protein E from coronaviruses MERS-Cov-2 and SARS-CoV-2 peptides with known experimental secondary structures in membrane bilayers. The two influenza-derived peptides are significantly different in the peptide sequence and secondary structure and more polar than the two coronavirus-derived peptides. Through a total of more than 50 µs of simulation time that could be accomplished in trifluoroethanol (TFE), as a membrane model, we characterized comparatively the folding behavior, helical stability, and helical propensity of these transmembrane peptides that match perfectly their experimental secondary structures, and we identified common motifs that reflect their quaternary organization and known (or not) biochemical function. We showed that BM2 is organized into two structurally distinct parts: a significantly more stable N-terminal half, and a fast-converting C-terminal half that continuously folds and unfolds between α-helical structures and non-canonical structures, which are mostly turns. In AM2, both the N-terminal half and C-terminal half are very flexible. In contrast, the two coronavirus-derived transmembrane peptides are much more stable and fast helix-formers when compared with the influenza ones. In particular, the SARS-derived peptide E appears to be the fastest and most stable helix-former of all the four viral peptides studied, with a helical structure that persists almost without disruption for the whole of its 10 µs simulation. By comparing the results with experimental observations, we benchmarked TFE in studying the conformation of membrane and hydrophobic peptides. This work provided accurate results suggesting a methodology to run long MD simulations and predict structural properties of biologically important membrane peptides.

On the presence of short-range periodicities in protein structures that are not related to established secondary structure elements.

Standard secondary structure elements such as α-helices or ß-sheets, are characterized by repeating backbone torsion angles (φ,ψ) at the single residue level. Two-residue motifs of the type (φ,ψ)2 are also observed in nonlinear conformations, mainly turns. Taking these observations a step further, it can be argued that there is no a priori reason why the presence of higher order periodicities can not be envisioned in protein structures, such as, for example, periodic transitions between successive residues of the type (…-α-ß-α-ß-α-…), or (…-ß-αL -ß-αL -ß-…), or (…-α-ß-αL -α-ß-αL -…), and so forth, where the symbols (α,ß,αL ) refer to the established Ramachandran-based residue conformations. From all such possible higher order periodicities, here we examine the deposited (with the PDB) protein structures for the presence of short-range periodical conformations comprising five consecutive residues alternating between two (and only two) distinct Ramachandran regions, for example, conformations of the type (α-ß-α-ß-α) or (ß-αL -ß-αL -ß), and so forth. Using a probabilistic approach, we have located several thousands of such peptapeptides, and these were clustered and analyzed in terms of their structural characteristics, their sequences, and their putative functional correlations using a gene ontology-based approach. We show that such nonstandard short-range periodicities are present in a large and functionally diverse sample of proteins, and can be grouped into two structurally conserved major types. Examination of the structural context in which these peptapeptides are observed gave no conclusive evidence for the presence of a persistent structural or functional role of these higher order periodic conformations.

Molecular simulation of peptides coming of age: Accurate prediction of folding, dynamics and structures.

The application of molecular dynamics simulations to study the folding and dynamics of peptides has attracted a lot of interest in the last couple of decades. Following the successful prediction of the folding of several proteins using molecular simulation, foldable peptides emerged as a favourable system mainly due to their application in improving protein structure prediction methods and in drug design studies. However, their performance is inherently linked to the accuracy of the empirical force fields used in the simulations, whose optimisation and validation is of paramount importance. Here we review the most important findings in the field of molecular peptide simulations and highlight the significant advancements made over the last twenty years. Special reference is made on the simulation of disordered peptides and the remaining challenge to find a force field able to describe accurately their conformational landscape.

Unusual α-Carbon Hydroxylation of Proline Promotes Active-Site Maturation.

The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexplored in bacteria. We describe here a so far unknown Pro hydroxylation activity which occurs in active sites of polysaccharide deacetylases (PDAs) from bacterial pathogens, modifying the protein backbone at the Cα atom of a Pro residue to produce 2-hydroxyproline (2-Hyp). This process modifies with high specificity a conserved Pro, shares with the deacetylation reaction the same active site and one catalytic residue, and utilizes molecular oxygen as source for the hydroxyl group oxygen of 2-Hyp. By providing additional hydrogen-bonding capacity, the Pro→2-Hyp conversion alters the active site and enhances significantly deacetylase activity, probably by creating a more favorable environment for transition-state stabilization. Our results classify this process as an active-site "maturation", which is highly atypical in being a protein backbone-modifying activity, rather than a side-chain-modifying one.

Sensitivity of Folding Molecular Dynamics Simulations to Even Minor Force Field Changes.

We examine the sensitivity of folding molecular dynamics simulations on the choice between three variants of the same force field (the AMBER99SB force field and its ILDN, NMR-ILDN, and STAR-ILDN variants). Using two different peptide systems (a marginally stable helical peptide and a ß-hairpin) and a grand total of more than 20 µs of simulation time we show that even relatively minor force field changes can lead to appreciable differences in the peptide folding behavior.

On the application of Good-Turing statistics to quantify convergence of biomolecular simulations.

Quantifying convergence and sufficient sampling of macromolecular molecular dynamics simulations is more often than not a source of controversy (and of various ad hoc solutions) in the field. Clearly, the only reasonable, consistent, and satisfying way to infer convergence (or otherwise) of a molecular dynamics trajectory must be based on probability theory. Ideally, the question we would wish to answer is the following: "What is the probability that a molecular configuration important for the analysis in hand has not yet been observed ?". Here we propose a method for answering a variant of this question by using the Good-Turing formalism for frequency estimation of unobserved species in a sample. Although several approaches may be followed in order to deal with the problem of discretizing the configurational space, for this work we use the classical RMSD matrix as a means to answering the following question: "What is the probability that a molecular configuration with an RMSD (from all other already observed configurations) higher than a given threshold has not actually been observed ?". We apply the proposed method to several different trajectories and show that the procedure appears to be both computationally stable and internally consistent. A free, open-source program implementing these ideas is immediately available for download via public repositories.

Structure determination through homology modelling and torsion-angle simulated annealing: application to a polysaccharide deacetylase from Bacillus cereus.

The structure of BC0361, a polysaccharide deacetylase from Bacillus cereus, has been determined using an unconventional molecular-replacement procedure. Tens of putative models of the C-terminal domain of the protein were constructed using a multitude of homology-modelling algorithms, and these were tested for the presence of signal in molecular-replacement calculations. Of these, only the model calculated by the SAM-T08 server gave a consistent and convincing solution, but the resulting model was too inaccurate to allow phase determination to proceed to completion. The application of slow-cooling torsion-angle simulated annealing (started from a very high temperature) drastically improved this initial model to the point of allowing phasing through cycles of model building and refinement to be initiated. The structure of the protein is presented with emphasis on the presence of a C(α)-modified proline at its active site, which was modelled as an α-hydroxy-L-proline.

Grcarma: A fully automated task-oriented interface for the analysis of molecular dynamics trajectories.

We report the availability of grcarma, a program encoding for a fully automated set of tasks aiming to simplify the analysis of molecular dynamics trajectories of biological macromolecules. It is a cross-platform, Perl/Tk-based front-end to the program carma and is designed to facilitate the needs of the novice as well as those of the expert user, while at the same time maintaining a user-friendly and intuitive design. Particular emphasis was given to the automation of several tedious tasks, such as extraction of clusters of structures based on dihedral and Cartesian principal component analysis, secondary structure analysis, calculation and display of root-meansquare deviation (RMSD) matrices, calculation of entropy, calculation and analysis of variance­covariance matrices, calculation of the fraction of native contacts, etc. The program is free-open source software available immediately for download.

Three force fields' views of the 3(10) helix.

Slowly but steadily bibliographic evidence is accumulating that the apparent convergence of the various biomolecular force fields as evidenced from simulations of proteins in the folded state does not hold true for folding simulations. Here we add one more example to the growing list of peptides and proteins for which different force fields show irreconcilable differences in their folding predictions, even at such a fundamental level as that of a peptide's secondary structure. We show that for an undecamer peptide that is known from two independent NMR structure determinations to have a mainly 3(10)-helical structure in solution, three mainstream biomolecular force fields give completely disparate predictions: The CHARMM force field (with the CMAP correction) predicts an outstandingly stable α-helical structure, in disagreement not only with the experimental structures, but also with experimental evidence obtained from circular dichroism. OPLS-AA shows an almost totally disordered peptide with the most frequently observed folded conformation corresponding to a ß-hairpin-like structure, again in disagreement with all available experimental evidence. Only the AMBER99SB force field appears to qualitatively agree with not only the general structural characteristics of the peptide (on the account of both NMR- and CD-based experiments), but to also correctly predict some of the experimentally observed interactions at the level of side chains. Possible interpretations of these findings are discussed.

On the application of structure-specific bulk-solvent models.

It is often discussed, mainly in connection with the rather high macromolecular R factors, that the treatment of bulk solvent in macromolecular refinement may lack the detail needed for modelling the solvent environment of molecules as complex as proteins and nucleic acids. This line of thought directly leads to the hypothesis that improvements in the modelling of the bulk solvent may substantially improve the agreement between the experimental data and the crystallographic models. Here, part of this hypothesis is being tested through the construction, via molecular-dynamics simulations, of a highly detailed, physics-based, structure-specific and crystallographic data-agnostic model of the bulk solvent of a known crystal structure. The water-distribution map obtained from the simulation is converted (after imposing space-group symmetry) to a constant (but scalable) partial structure factor which is then added in a re-refinement of the crystal structure. Compared with the simple Babinet-based correction, a reduction of the totally cross-validated free R value by 0.3% is observed. The implications and possible interpretations of these results are discussed.

A molecular dynamics simulation study on the propensity of Asn-Gly-containing heptapeptides towards ß-turn structures: Comparison with ab initio quantum mechanical calculations.

Both molecular mechanical and quantum mechanical calculations play an important role in describing the behavior and structure of molecules. In this work, we compare for the same peptide systems the results obtained from folding molecular dynamics simulations with previously reported results from quantum mechanical calculations. More specifically, three molecular dynamics simulations of 5 µs each in explicit water solvent were carried out for three Asn-Gly-containing heptapeptides, in order to study their folding and dynamics. Previous data, based on quantum mechanical calculations within the DFT framework have shown that these peptides adopt ß-turn structures in aqueous solution, with type I' ß-turn being the most preferred motif. The results from our analyses indicate that at least for the given systems, force field and simulation protocol, the two methods diverge in their predictions. The possibility of a force field-dependent deficiency is examined as a possible source of the observed discrepancy.

Catalytic activity regulation through post-translational modification: the expanding universe of protein diversity.

Protein composition is restricted by the genetic code to a relatively small number of natural amino acids. Similarly, the known three-dimensional structures adopt a limited number of protein folds. However, proteins exert a large variety of functions and show a remarkable ability for regulation and immediate response to intracellular and extracellular stimuli. To some degree, the wide variability of protein function can be attributed to the post-translational modifications. Post-translational modifications have been observed in all kingdoms of life and give to proteins a significant degree of chemical and consequently functional and structural diversity. Their importance is partly reflected in the large number of genes dedicated to their regulation. So far, hundreds of post-translational modifications have been observed while it is believed that many more are to be discovered along with the technological advances in sequencing, proteomics, mass spectrometry and structural biology. Indeed, the number of studies which report novel post translational modifications is getting larger supporting the notion that their space is still largely unexplored. In this review we explore the impact of post-translational modifications on protein structure and function with emphasis on catalytic activity regulation. We present examples of proteins and protein families whose catalytic activity is substantially affected by the presence of post translational modifications and we describe the molecular basis which underlies the regulation of the protein function through these modifications. When available, we also summarize the current state of knowledge on the mechanisms which introduce these modifications to protein sites.

On the quaternary association of the type III secretion system HrcQB-C protein: experimental evidence differentiates among the various oligomerization models.

The HrcQB protein from the plant pathogen Pseudomonas syringae is a core component of the bacterial type III secretion apparatus. The core consists of nine proteins widely conserved among animal and plant pathogens which also share sequence and structural similarities with proteins from the bacterial flagellum. Previous studies of the carboxy-terminal domain of HrcQB (HrcQB-C) and its flagellar homologue, FliN-C, have revealed extensive sequence and structural homologies, similar subcellular localization, and participation in analogous protein-protein interaction networks. It is not clear however whether the similarities between the two proteins extend to the level of quaternary association which is essential for the formation of higher-order structures within the TTSS. Even though the crystal structure of the FliN is a dimer, more detailed studies support a tetrameric donut-like association. However, both models, dimer and donut-like tetramer, are quite different from the crystallographic elongated dimer of dimers of the HrcQB-C. To resolve this discrepancy we performed a multidisciplinary investigation of the quaternary association of the HrcQB-C, including mass-spectrometry, electrophoresis in non-reductive conditions, gel filtration, glutaraldehyde cross-linking and small angle X-ray scattering. Our experiments indicate that stable tetramers of elongated shape are assembled in solution, in agreement with the results of crystallographic studies. Circular dichroism data are consistent with a dimer-dimer interface analogous to the one established in the crystal structure. Finally, molecular dynamics simulations reveal the relative orientation of the dimers forming the tetramers and the possible differences from that of the crystal structure.

Folding Molecular Dynamics Simulation of a gp41-Derived Peptide Reconcile Divergent Structure Determinations.

T-20 peptide is the first FDA-approved fusion inhibitor against AIDS/HIV-1 gp41 protein. Part of it, the gp41[659-671] peptide, that contains the complete epitope for the neutralizing 2F5 monoclonal antibody, has been found experimentally in a number of divergent structures. Herein, we attempt to reconcile them by using unbiased large-scale all-atom molecular dynamics folding simulations. We show that our approach can successfully capture the peptide's heterogeneity and reach each and every experimentally determined conformation in sub-angstrom accuracy, whilst preserving the peptide's disordered nature. Our analysis also unveils that the minor refinements within the AMBER99SB family of force fields can lead to appreciable differences in the predicted conformational stability arising from subtle differences in the helical- and ß-region of the Ramachandran plot. Our work underlines the contribution of molecular dynamics simulation in structurally characterizing pharmacologically important peptides of ambiguous structure.

Folding Simulations of a Nuclear Receptor Box-Containing Peptide Demonstrate the Structural Persistence of the LxxLL Motif Even in the Absence of Its Cognate Receptor.

Regulation of nuclear receptors by their coactivators involves the recognition and binding of a specific sequence motif contained in the coactivator sequence. This motif is known as the nuclear receptor (NR) box and contains a conserved LxxLL subsequence, where L is leucine and x is any amino acid residue. Crystallographic studies have shown that the LxxLL motifs adopt an α-helical conformation when bound to their cognate nuclear receptors. Here we use an extensive set of folding molecular dynamics simulations to examine whether the α-helical conformation demonstrated by the LxxLL motifs in the bound state may represent a persistent structural preference of these peptides even in the absence of their cognate receptors. To this end, we have performed a grand total of 35 µs of adaptive tempering folding simulations of an NR-box-containing peptide derived from Drosophila's fushi tarazu segmentation gene product. Our simulations-performed using full electrostatics and an explicit representation of two different solvents (water and a TFE/water mixture)-clearly indicate the presence of a persistent helical preference of the LxxLL motif with a concomitant native-like structure and contacts between the motif's leucine residues. To lend further support to our findings, we compare the simulation-derived peptide dynamics with experimental NMR-derived nuclear Overhauser effect (NOE) measurements that had been previously obtained for the same peptide in the same two solvents. The comparison demonstrates a quantitative agreement between simulation and experiment with average upper bound NOE violations of less than 0.084 Å, thus independently validating our main conclusion concerning the intrinsic preference of NR-box motifs to form helical structures even in the absence of their cognate receptors.

Crystal structure of the BcZBP, a zinc-binding protein from Bacillus cereus.

Bacillus cereus is an opportunistic pathogenic bacterium closely related to Bacillus anthracis, the causative agent of anthrax in mammals. A significant portion of the B. cereus chromosomal genes are common to B. anthracis, including genes which in B. anthracis code for putative virulence and surface proteins. B. cereus thus provides a convenient model organism for studying proteins potentially associated with the pathogenicity of the highly infectious B. anthracis. The zinc-binding protein of B. cereus, BcZBP, is encoded from the bc1534 gene which has three homologues to B. anthracis. The protein exhibits deacetylase activity with the N-acetyl moiety of the N-acetylglucosamine and the diacetylchitobiose and triacetylchitotriose. However, neither the specific substrate of the BcZBP nor the biochemical pathway have been conclusively identified. Here, we present the crystal structure of BcZBP at 1.8 A resolution. The N-terminal part of the 234 amino acid protein adopts a Rossmann fold whereas the C-terminal part consists of two beta-strands and two alpha-helices. In the crystal, the protein forms a compact hexamer, in agreement with solution data. A zinc binding site and a potential active site have been identified in each monomer. These sites have extensive similarities to those found in two known zinc-dependent hydrolases with deacetylase activity, MshB and LpxC, despite a low degree of amino acid sequence identity. The functional implications and a possible catalytic mechanism are discussed.

Characterizing a partially ordered miniprotein through folding molecular dynamics simulations: Comparison with the experimental data.

The villin headpiece helical subdomain (HP36) is one of the best known model systems for computational studies of fast-folding all-α miniproteins. HP21 is a peptide fragment-derived from HP36-comprising only the first and second helices of the full domain. Experimental studies showed that although HP21 is mostly unfolded in solution, it does maintain some persistent native-like structure as indicated by the analysis of NMR-derived chemical shifts. Here we compare the experimental data for HP21 with the results obtained from a 15-µs long folding molecular dynamics simulation performed in explicit water and with full electrostatics. We find that the simulation is in good agreement with the experiment and faithfully reproduces the major experimental findings, namely that (a) HP21 is disordered in solution with <10% of the trajectory corresponding to transiently stable structures, (b) the most highly populated conformer is a native-like structure with an RMSD from the corresponding portion of the HP36 crystal structure of <1 Å, (c) the simulation-derived chemical shifts-over the whole length of the trajectory-are in reasonable agreement with the experiment giving reduced χ(2) values of 1.6, 1.4, and 0.8 for the Δδ(13) C(α) , Δδ(13) CO, and Δδ(13) C(ß) secondary shifts, respectively (becoming 0.8, 0.7, and 0.3 when only the major peptide conformer is considered), and finally, (d) the secondary structure propensity scores are in very good agreement with the experiment and clearly indicate the higher stability of the first helix. We conclude that folding molecular dynamics simulations can be a useful tool for the structural characterization of even marginally stable peptides.

Folding molecular dynamics simulations accurately predict the effect of mutations on the stability and structure of a vammin-derived peptide.

Folding molecular dynamics simulations amounting to a grand total of 4 µs of simulation time were performed on two peptides (with native and mutated sequences) derived from loop 3 of the vammin protein and the results compared with the experimentally known peptide stabilities and structures. The simulations faithfully and accurately reproduce the major experimental findings and show that (a) the native peptide is mostly disordered in solution, (b) the mutant peptide has a well-defined and stable structure, and (c) the structure of the mutant is an irregular ß-hairpin with a non-glycine ß-bulge, in excellent agreement with the peptide's known NMR structure. Additionally, the simulations also predict the presence of a very small ß-hairpin-like population for the native peptide but surprisingly indicate that this population is structurally more similar to the structure of the native peptide as observed in the vammin protein than to the NMR structure of the isolated mutant peptide. We conclude that, at least for the given system, force field, and simulation protocol, folding molecular dynamics simulations appear to be successful in reproducing the experimentally accessible physical reality to a satisfactory level of detail and accuracy.