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.

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 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 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.

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.

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.

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.

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.

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.

Pinda: a web service for detection and analysis of intraspecies gene duplication events.

We present Pinda, a Web service for the detection and analysis of possible duplications of a given protein or DNA sequence within a source species. Pinda fully automates the whole gene duplication detection procedure, from performing the initial similarity searches, to generating the multiple sequence alignments and the corresponding phylogenetic trees, to bootstrapping the trees and producing a Z-score-based list of duplication candidates for the input sequence. Pinda has been cross-validated using an extensive set of known and bibliographically characterized duplication events. The service facilitates the automatic and dependable identification of gene duplication events, using some of the most successful bioinformatics software to perform an extensive analysis protocol. Pinda will prove of use for the analysis of newly discovered genes and proteins, thus also assisting the study of recently sequenced genomes. The service's location is http://orion.mbg.duth.gr/Pinda. The source code is freely available via https://github.com/dgkontopoulos/Pinda/.

On the foldability of tryptophan-containing tetra- and pentapeptides: an exhaustive molecular dynamics study.

Short peptides serve as minimal model systems to decipher the determinants of foldability due to their simplicity arising from their smaller size, their ability to echo protein-like structural characteristics, and their direct implication in force field validation. Here, we describe an effort to identify small peptides that can still form stable structures in aqueous solutions. We followed the in silico folding of a selected set of 8640 tryptophan-containing tetra- and pentapeptides through 15 210 molecular dynamics simulations amounting to a total of 272.46 µs using explicit representation of the solute and full treatment of the electrostatics. The evaluation and sorting of peptides is achieved through scoring functions, which include terms based on interatomic vector distances, atomic fluctuations, and rmsd matrices between successive frames of a trajectory. Highly scored peptides are studied further via successive simulation rounds of increasing simulation length and using different empirical force fields. Our method suggested only a handful of peptides with strong foldability prognosis. The discrepancies between the predictions of the various force fields for such short sequences are also extensively discussed. We conclude that the vast majority of such short peptides do not adopt significantly stable structures in water solutions, at least based on our computational predictions. The present work can be utilized in the rational design and engineering of bioactive peptides with desired molecular properties.

As good as it gets? Folding molecular dynamics simulations of the LytA choline-binding peptide result to an exceptionally accurate model of the peptide structure.

Folding simulations of a choline-binding peptide derived from the Streptococcus pneumoniae LytA protein converged to a model of the peptide's folded state structure which is in outstanding agreement with the experimentally-determined structures, reaching values for the root mean squared deviation as low as 0.24Å for the peptide's backbone atoms and 0.65Å for all non-hydrogen atoms.

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.

Using J-coupling constants for force field validation: application to hepta-alanine.

A computational solution to the protein folding problem is the holy grail of biomolecular simulation and of the corresponding force fields. The complexity of the systems used for folding simulations precludes a direct feedback between the simulations and the force fields, thus necessitating the study of simpler systems with sufficient experimental data to allow force field optimization and validation. Recent studies on short polyalanine peptides of increasing length (up to penta-alanine) indicated the presence of a systematic deviation between the experimental (NMR-derived) J-couplings and the great majority of biomolecular force fields, with the χ(2) values for even the best-performing force fields being in the 1.4-1.8 range. Here we show that by increasing the number of residues to seven and by achieving convergence through an increase of the simulation time to 2 µs, we can identify one force field (the AMBER99SB force field, out of the three force fields studied) which when compared with the experimental J-coupling data (and for a specific set of Karplus equation parameters and estimated J-coupling errors previously used in the literature) gave a value of χ(2) = 0.99, indicating that full statistical consistency between experiment and simulation is feasible. However, and as a detailed analysis of the effects of estimated errors shows, the χ(2) values may be unsuitable as indicators of the goodness of fit of the various biomolecular force fields.

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.