Exploring ssDNA translocation through α-hemolysin using coarse-grained steered molecular dynamics.

Protein nanopores have proven to be effective for single-molecule studies, particularly for single-stranded DNA (ssDNA) translocation. Previous experiments demonstrated their ability to distinguish differences in purine and pyrimidine bases and in the orientation of the ssDNA molecule inside nanopores. Unfortunately, the microscopic details of ssDNA translocation over experimental time scales, which are not accessible through all-atom molecular dynamics (MD), have yet to be examined. However, coarse-grained (CG) MD simulations enable systems to be simulated over longer characteristic times closer to experiments than all-atom MD. This paper studies ssDNA translocation through α-hemolysin nanopores exploiting steered MD using the MARTINI CG force field. The impacts of the sequence length, orientation inside the nanopore and DNA charges on translocation dynamics as well as the conformational dynamics of ssDNA during the translocation are explored. Our results highlight the efficacy of CG molecular dynamics in capturing the experimental properties of ssDNA translocation, including a wide distribution in translocation times per base. In particular, the phosphate charges of the DNA molecule are crucial in the translocation dynamics and impact the translocation rate. Additionally, the influence of the ssDNA molecule orientation on the translocation rate is explained by the conformational differences of ssDNA inside the nanopore during its translocation. Our study emphasizes the significance of obtaining sufficient statistics via CG MD, which can elucidate the great variety of translocation processes.

Current Rectification and Ionic Selectivity of α-Hemolysin: Coarse-Grained Molecular Dynamics Simulations.

In order to understand the physical processes of nanopore experiments at the molecular level, microscopic information from molecular dynamics is greatly needed. Coarse-grained models are a good alternative to classical all-atom models since they allow longer and faster simulations. We performed coarse-grained molecular dynamics of the ionic transport through the α-hemolysin protein nanopore, inserted into a lipid bilayer surrounded by solvent and ions. For this purpose, we used the MARTINI coarse-grained force field and its polarizable water solvent (PW). Moreover, the electric potential difference applied experimentally was mimicked by the application of an electric field to the system. We present, in this study, the results of 1.5 µs long-molecular dynamics simulations of 12 different systems for which different charged amino acids were neutralized, each of them in the presence of nine different electric fields ranging between ±0.04 V/nm (a total of around 100 simulations). We were able to observe several specific features of this pore, current asymmetry and anion selectivity, in agreement with previous studies and experiments, and we identified the charged amino acids responsible for these current behaviors, therefore validating our coarse-grain approach to study ionic transport through nanopores. We also propose a microscopic explanation of these ionic current features using ionic density maps.

Ionic transport through a protein nanopore: a Coarse-Grained Molecular Dynamics Study.

The MARTINI coarse-grained (CG) force field is used to test the ability of CG models to simulate ionic transport through protein nanopores. The ionic conductivity of CG ions in solution was computed and compared with experimental results. Next, we studied the electrostatic behavior of a solvated CG lipid bilayer in salt solution under an external electric field. We showed this approach correctly describes the experimental conditions under a potential bias. Finally, we performed CG molecular dynamics simulations of the ionic transport through a protein nanopore (α-hemolysin) inserted in a lipid bilayer, under different electric fields, for 2-3 microseconds. The resulting I - V curve is qualitatively consistent with experiments, although the computed current is one order of magnitude smaller. Current saturation was observed for potential biases over ±350 mV. We also discuss the time to reach a stationary regime and the role of the protein flexibility in our CG simulations.

Interaction of chemokine receptor CXCR4 in monomeric and dimeric state with its endogenous ligand CXCL12: coarse-grained simulations identify differences.

Despite the recent resolutions of the crystal structure of the chemokine receptor CXCR4 in complex with small antagonists or viral chemokine, a description at the molecular level of the interactions between the full-length CXCR4 and its endogenous ligand, the chemokine CXCL12, in relationship with the receptor recognition and activation, is not yet completely elucidated. Moreover, since CXCR4 is able to form dimers, the question of whether the CXCR4-CXCL12 complex has a 1:1 or 2:1 preferential stoichiometry is still an open question. We present here results of coarse-grained protein-protein docking and molecular dynamics simulations of CXCL12 in association with CXCR4 in monomeric and dimeric states. Our proposed models for the 1:1 and 2:1 CXCR4-CXCL12 quaternary structures are consistent with recognition and activation motifs of both partners provided by the available site-directed mutagenesis data. Notably, we observed that in the 2:1 complex, the chemokine N-terminus makes more steady contacts with the receptor residues critical for binding and activation than in the 1:1 structure, suggesting that the 2:1 stoichiometry would favor the receptor signaling activity with respect to the 1:1 association.

Structure of ring-shaped Aß42 oligomers determined by conformational selection.

The oligomerization of amyloid beta (Aß) peptides into soluble non-fibrillar species plays a critical role in the pathogenesis of Alzheimer's disease. However, it has been challenging to characterize the tertiary and quaternary structures of Aß peptides due to their disordered nature and high aggregation propensity. In this work, replica exchange molecular dynamics simulations were used to explore the conformational space of Aß42 monomer. Among the most populated transient states, we identified a particular conformation which was able to generate ring-shaped pentamers and hexamers, when docked onto itself. The structures of these aggregates were stable during microsecond all-atom MD simulations in explicit solvent. In addition to high resolution models of these oligomers, this study provides support for the conformational selection mechanism of Aß peptide self-assembly.

Influence of GTP/GDP and magnesium ion on the solvated structure of the protein FtsZ: a molecular dynamics study.

We present here a structural analysis of ten extensive all-atom molecular dynamics simulations of the monomeric protein FtsZ in various binding states. Since the polymerization and GTPase activities of FtsZ depend on the nature of a bound nucleotide as well as on the presence of a magnesium ion, we studied the structural differences between the average conformations of the following five systems: FtsZ-Apo, FtsZ-GTP, FtsZ-GDP, FtsZ-GTP-Mg, and FtsZ-GDP-Mg. The in silico solvated average structure of FtsZ-Apo significantly differs from the crystallographic structure 1W59 of FtsZ which was crystallized in a dimeric form without nucleotide and magnesium. The simulated Apo form of the protein also clearly differs from the FtsZ structures when it is bound to its ligand, the most important discrepancies being located in the loops surrounding the nucleotide binding pocket. The three average structures of FtsZ-GTP, FtsZ-GDP, and FtsZ-GTP-Mg are overall similar, except for the loop T7 located at the opposite side of the binding pocket and whose conformation in FtsZ-GDP notably differs from the one in FtsZ-GTP and FtsZ-GTP-Mg. The presence of a magnesium ion in the binding pocket has no impact on the FtsZ conformation when it is bound to GTP. In contrast, when the protein is bound to GDP, the divalent cation causes a translation of the nucleotide outwards the pocket, inducing a significant conformational change of the loop H6-H7 and the top of helix H7.

Modeling Protein-Protein Recognition in Solution Using the Coarse-Grained Force Field SCORPION.

We present here the SCORPION-Solvated COaRse-grained Protein interactION-force field, a physics-based simplified coarse-grained (CG) force field. It combines our previous CG protein model and a novel particle-based water model which makes it suitable for Molecular Dynamics (MD) simulations of protein association processes. The protein model in SCORPION represents each amino acid with one to three beads, for which electrostatic and van der Waals effective interactions are fitted separately to reproduce those of the all-atom AMBER force field. The protein internal flexibility is accounted for by an elastic network model (ENM). We now include in SCORPION a new Polarizable Coarse-Grained Solvent (PCGS) model, which is computationally efficient, consistent with the protein CG representation, and yields accurate electrostatic free energies of proteins. SCORPION is used here for the first time to perform hundreds-of-nanoseconds-long MD simulations of protein/protein recognition in water, here the case of the barnase/barstar complex. These MD simulations showed that, for five of a total of seven simulations starting from several initial conformations, and after a time going from 1 to 500 ns, the proteins bind in a conformation very close to the native bound structure and remain stable in this conformation for the rest of the simulation. An energetic analysis of these MD show that this recognition is driven both by van der Waals and electrostatic interactions between proteins. SCORPION appears therefore as a useful tool to study protein-protein recognition in a solvated environment.

A coarse-grained protein-protein potential derived from an all-atom force field.

In order to study protein-protein nonbonded interactions, we present the development of a new reduced protein model that represents each amino acid residue with one to three coarse grains, whose physical properties are derived in a consistent bottom-up procedure from the higher-resolution all-atom AMBER force field. The resulting potential energy function is pairwise additive and includes distinct van-der-Waals and Coulombic terms. The van-der-Waals effective interactions are deduced from preliminary molecular dynamics simulations of all possible amino acid homodimers. They are best represented by a soft 1/r6 repulsion and a Gaussian attraction, with parameters obeying Lorentz-Berthelot mixing rules. For the Coulombic interaction, coarse grain charges are optimized for each separate protein in order to best represent the all-atom electrostatic potential outside the protein core. This approach leaves the possibility of using any implicit solvent model to describe solvation effects and electrostatic screening. The coarse-grained force field is tested carefully for a small homodimeric complex, the magainin. It is shown to reproduce satisfactorily the specificity of the all-atom underlying potential, in particular within a PB/SA solvation model. The coarse-grained potential is applied to the redocking prediction of three different protein-protein complexes: the magainin dimer, the barnase-barstar, and the trypsin-BPTI complexes. It is shown to provide per se an efficient and discriminating scoring energy function for the protein-protein docking problem that remains pertinent at both the global and refinement stage.

Thermodynamic basis for promiscuity and selectivity in protein-protein interactions: PDZ domains, a case study.

Like other protein-protein interaction domains, PDZ domains are involved in many key cellular processes. These processes often require that specific multiprotein complexes be assembled, a task that PDZ domains accomplish by binding to specific peptide motifs in target proteins. However, a growing number of experimental studies show that PDZ domains (like other protein-protein interaction domains) can engage in a variety of interactions and bind distinct peptide motifs. Such promiscuity in ligand recognition raises intriguing questions about the molecular and thermodynamic mechanisms that can sustain it. To identify possible sources of promiscuity and selectivity underlying PDZ domain interactions, we performed molecular dynamics simulations of 20 to 25 ns on a set of 12 different PDZ domain complexes (for the proteins PSD-95, Syntenin, Erbin, GRIP, NHERF, Inad, Dishevelled, and Shank). The electrostatic, nonpolar, and configurational entropy binding contributions were evaluated using the MM/PBSA method combined with a quasi-harmonic analysis. The results revealed that PDZ domain interactions are characterized by overwhelmingly favorable nonpolar contributions and almost negligible electrostatic components, a mix that may readily sustain promiscuity. In addition, despite the structural similarity in fold and in recognition modes, the entropic and other dynamical aspects of binding were remarkably variable not only across PDZ domains but also for the same PDZ domain bound to distinct ligands. This variability suggests that entropic and dynamical components can play a role in determining selectivity either of PDZ domain interactions with peptide ligands or of PDZ domain complexes with downstream effectors.

Particle-Based Implicit Solvent Model for Biosimulations: Application to Proteins and Nucleic Acids Hydration.

In addition to the simulation of two proteins described previously, we report on the application of our recently developed particle-based implicit solvent model to the simulations of four nucleic acid molecules, the 17 bases anticodon hairpin of the Asp-tRNA, the decamer d(CCGCCGGCGG) in both A and B form, and the containing EcoRI restriction site dodecamer d(CGCGAATTCGCG). The solvent is represented by a fluid of Lennard-Jones polarizable pseudoparticles of molecular size, the induced dipoles of which are sensitive to the solute electric field but not to each other. When implemented in a molecular dynamics algorithm with the Amber94 force field, the model allows to simulate efficiently the conformational evolution of the nucleic acids, yielding stable three-dimensional structures in agreement with experiments and other simulations in explicit solvent. In the same run, it is also able to provide estimations of the electrostatic solvation free energy within short time windows which correlate well with the Poisson-Boltzmann calculations. In addition, the molecular aspect of the solvent model allows for the reproduction of the highly localized water molecules in the major or minor grooves of the nucleic acid double helices, despite the absence of explicit water hydrogen bonds.

A semi-implicit solvent model for the simulation of peptides and proteins.

We present a new model of biomolecules hydration based on macroscopic electrostatic theory, that can both describe the microscopic details of solvent-solute interactions and allow for an efficient evaluation of the electrostatic hydration free energy. This semi-implicit model considers the solvent as an ensemble of polarizable pseudoparticles whose induced dipole describe both the electronic and orientational solvent polarization. In the presented version of the model, there is no mutual dipolar interaction between the particles, and they only interact through short-ranged Lennard-Jones interactions. The model has been integrated into a molecular dynamics code, and offers the possibility to simulate efficiently the conformational evolution of biomolecules. It is able to provide estimations of the electrostatic solvation free energy within short time windows during the simulation. It has been applied to the study of two small peptides, the octaalanine and the N-terminal helix of ribonuclease A, and two proteins, the bovine pancreatic trypsin inhibitor and the B1 immunoglobin-binding domain of streptococcal protein G. Molecular dynamics simulations of these biomolecules, using a slightly modified Amber force field, provide stable and meaningful trajectories in overall agreement with experiments and all-atom simulations. Correlations with respect to Poisson-Boltzmann electrostatic solvation free energies are also presented to discuss the parameterization of the model and its consequences.