Calculation of protein ionization equilibria with conformational sampling: pK(a) of a model leucine zipper, GCN4 and barnase.

The use of conformational ensembles provided by nuclear magnetic resonance (NMR) experiments or generated by molecular dynamics (MD) simulations has been regarded as a useful approach to account for protein motions in the context of pK(a) calculations, yet the idea has been tested occasionally. This is the first report of systematic comparison of pK(a) estimates computed from long multiple MD simulations and NMR ensembles. As model systems, a synthetic leucine zipper, the naturally occurring coiled coil GCN4, and barnase were used. A variety of conformational averaging and titration curve-averaging techniques, or combination thereof, was adopted and/or modified to investigate the effect of extensive global conformational sampling on the accuracy of pK(a) calculations. Clustering of coordinates is proposed as an approach to reduce the vast diversity of MD ensembles to a few structures representative of the average electrostatic properties of the system in solution. Remarkable improvement of the accuracy of pK(a) predictions was achieved by the use of multiple MD simulations. By using multiple trajectories the absolute error in pK(a) predictions for the model leucine zipper was reduced to as low as approximately 0.25 pK(a) units. The validity, advantages, and limitations of explicit conformational sampling by MD, compared with the use of an average structure and a high internal protein dielectric value as means to improve the accuracy of pK(a) calculations, are discussed.     

Kinetic analysis of molecular dynamics simulations reveals changes in the denatured state and switch of folding pathways upon single-point mutation of a beta-sheet miniprotein

The effects of a single-point mutation on folding thermodynamics and kinetics are usually interpreted by focusing on the native structure and the transition state. Here, the entire conformational spaces of a 20-residue three-stranded antiparallel beta-sheet peptide (double hairpin) and of its single-point mutant W10V are sampled close to the melting temperature by equilibrium folding-unfolding molecular dynamics simulations for a total of 40 micros. The folded state as well as the most populated free energy basins in the denatured state are isolated by grouping conformations according to fast relaxation at equilibrium. Such kinetic analysis provides more detailed and useful information than a simple projection of the free energy. The W10V mutant has the same native structure as the wild type peptide, and similar folding rate and stability. In the denatured state, the N-terminal hairpin is about 20% more structured in W10V than the wild type mainly because of van der Waals interactions. Notably, the W10V mutation influences also the van der Waals energy at the transition state ensemble causing a shift in the ratio of fluxes between two different transition state regions on parallel folding pathways corresponding to nucleation at either of the two beta-hairpins. Previous experimental studies have focused on the effects of denaturant-dependent or temperature-dependent changes in the structure of the denatured state. The atomistic simulations show that a single-point mutation in the central strand of a beta-sheet peptide results in remarkable changes in the topography of the denatured state ensemble. These changes modulate the relative accessibility of parallel folding pathways because of kinetic partitioning of the denatured state. Therefore, the observed dependence of the folding process on the starting ensemble raises questions on the biological significance of in vitro folding studies under strongly denaturing conditions.     

The folding and dimerization of HIV-1 protease: evidence for a stable monomer from simulations

HIV-1 protease (PR) is a major drug target in combating AIDS, as it plays a key role in maturation and replication of the virus. Six FDA-approved drugs are currently in clinical use, all designed to inhibit enzyme activity by blocking the active site, which exists only in the dimer. An alternative inhibition mode would be required to overcome the emergence of drug-resistance through the accumulation of mutations. This might involve inhibiting the formation of the dimer itself. Here, the folding of HIV-1 PR dimer is studied with several simulation models appropriate for folding mechanism studies. Simulations with an off-lattice Gō-model, which corresponds to a perfectly funneled energy landscape, indicate that the enzyme is formed by association of structured monomers. All-atom molecular dynamics simulations strongly support the stability of an isolated monomer. The conjunction of results from a model that focuses on the protein topology and a detailed all-atom force-field model suggests, in contradiction to some reported equilibrium denaturation experiments, that monomer folding and dimerization are decoupled. The simulation result is, however, in agreement with the recent NMR detection of folded monomers of HIV-1 PR mutants with a destabilized interface. Accordingly, the design of dimerization inhibitors should not focus only on the flexible N and C termini that constitute most of the dimer interface, but also on other structured regions of the monomer. In particular, the relatively high phi values for residues 23-35 and 79-87 in both the folding and binding transition states, together with their proximity to the interface, highlight them as good targets for inhibitor design.     

Unfolding transition state and intermediates of the tumor suppressor p16INK4a investigated by molecular dynamics simulations

The ankyrin repeat is one of the most common protein motifs and is involved in protein-protein interactions. It consists of 33 residues that assume a beta-hairpin helix-loop-helix fold. Mutagenesis and kinetic experiments (Phi-value analysis of the folding transition state) have shown that the tumor suppressor p16(INK4a), a four-repeat protein, unfolds sequentially starting from the two N-terminal repeats. Here, the flexibility of p16(INK4a) at room temperature and its unfolding mechanism at high temperature have been investigated by multiple molecular dynamics runs in explicit water for a total simulation time of 0.65 micros. The transition state ensemble (TSE) of p16(INK4a) was identified by monitoring both the deviation from the experimental Phi values and sudden conformational changes along the unfolding trajectories. Conformations in the TSE have a mainly unstructured second repeat whereas the other repeats are almost completely folded. A rigid-body displacement of the first repeat involving both a rotation and translation is observed in all molecular dynamics simulations at high temperature. The Trp(15), Pro(75), and Ala(76) side-chains are more buried in the TSE than the native state. The sequential unfolding starting at the second repeat is in agreement with the mutagenesis studies whereas the displacement of the first repeat and the presence of nonnative interactions at the TSE are simulation results which supplement the experimental data. Furthermore, the unfolding trajectories reveal the presence of two on-pathway intermediates with partial alpha-helical structure. Finally, on the basis of the available experimental and simulation results we suggest that in modular proteins the shift of the folding TSE toward the native structure upon reduction of the number of tandem repeats is consistent with the Hammond effect.     

Computational models for the prediction of polypeptide aggregation propensity

In amyloid fibrils, beta-strand conformations of polypeptide chains, or segments thereof, are perpendicular to the fibril axis, but knowledge of their three dimensional structure at atomic level of detail is scarce. Two types of computational approaches have been developed recently for investigating the aggregation propensity of peptides and proteins and identifying the segments most prone to form fibrils (hot spots). The physicochemical properties of the natural amino acids (e.g. beta-propensity, hydrophobicity, aromatic content and charge) have been used to derive phenomenological models able to predict changes in aggregation rate upon mutation, as well as absolute rates and hot spots. Applications of these models to entire proteomes have provided evidence that intrinsically disordered proteins are less amyloidogenic than globular proteins. In the second type of approach, amyloidogenic polypeptides have been decomposed into overlapping segments, and atomistic simulations of three or more copies of each segment have been performed to obtain insights into aggregation propensity and structural details of the ordered aggregates (e.g. turn regions).     

Identification and characterisation of a novel adhesin Ifp in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

Background  

In order to identify new virulence determinants in Y. pseudotuberculosis a comparison between its genome and that of Yersinia pestis was undertaken. This reveals dozens of pseudogenes in Y. pestis, which are still putatively functional in Y. pseudotuberculosis and may be important in the enteric lifestyle. One such gene, YPTB1572 in the Y. pseudotuberculosis IP32953 genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic (EPEC) and enterohaemorraghic (EHEC) Escherichia coli.     

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature- and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5° higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.     

Monte Carlo docking of oligopeptides to proteins.

A new two-step procedure has been developed for the docking of flexible oligopeptide chains of unknown conformation to static proteins of known structure. In the first step positions and conformations are sampled and the association energy minimized starting from an approximate preselected docking position. The resulting conformations are further optimized in the second step by a Metropolis Monte Carlo minimization, which optimizes each of these structures. The method has been tested on the HIV-1 aspartic proteinase complex with an inhibitor, whose crystallographic structure is known at 2.3 A resolution. Furthermore, the application of this method to the docking of the hendecapeptide 58-68 of the influenza A virus matrix protein to the HLA-A2 molecule produced results which are in agreement with experimental observations in identifying side chains critical for T cell recognition and residues responsible of MHC protein binding.