Fabrizio Marinelli

PLUMED: a portable plugin for free-energy calculations with molecular dynamics

Here we present a program aimed at free-energy calculations in molecular systems. It consists of a series of routines that can be interfaced with the most popular classical molecular dynamics (MD) codes through a simple patching procedure. This leaves the possibility for the user to exploit many different MD engines depending on the system simulated and on the computational resources available. Free-energy calculations can be performed as a function of many collective variables, with a particular focus on biological problems, and using state-of-the-art methods such as metadynamics, umbrella sampling and Jarzynski-equation based steered MD. The present software, written in ANSI-C language, can be easily interfaced with both Fortran and C/C++ codes.

A Kinetic Model of Trp-Cage Folding from Multiple Biased Molecular Dynamics Simulations

Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins. Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala3-Nme peptide. For the Trp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap. Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at of 4.4 Å from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the and chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data.

Substrate Binding Mechanism of HIV-1 Protease from Explicit-Solvent Atomistic Simulations

The binding mechanism of a peptide substrate (Thr-Ile-Met-Met-Gln-Arg, cleavage site p2-NC of the viral polyprotein) to wild-type HIV-1 protease has been investigated by 1.6 micros biased all-atom molecular dynamics simulations in explicit water. The configuration space has been explored biasing seven reaction coordinates by the bias-exchange metadynamics technique. The structure of the Michaelis complex is obtained starting from the substrate outside the enzyme within a backbone rmsd of 0.9 A. The calculated free energy of binding is -6 kcal/mol, and the kinetic constants for association and dissociation are 1.3 x 10(6) M(-1) s(-1) and 57 s(-1), respectively, consistent with experiments. In the main binding pathway, the flaps of the protease do not open sizably. The substrate slides inside the enzyme cavity from the tight lateral channel. This may contrast with the natural polyprotein substrate which is expected to bind by opening the flaps. Thus, mutations might influence differently the binding kinetics of peptidomimetic ligands and of the natural substrate.

Targeting biomolecular flexibility with metadynamics.

Metadynamics calculations allow investigating structure, plasticity, and energetics in a variety of biological processes spanning from molecular docking to protein folding. Recent theoretical developments have led to applications to increasingly complex systems and processes stepping up the biological relevance of the problem solved. Here, after summarizing recent technical advances and applications, we give a perspective of the method as a tool for enzymology and for the prediction of NMR and other spectroscopic data.

METAGUI. A VMD interface for analyzing metadynamics and molecular dynamics simulations

We present a new computational tool, METAGUI, which extends the VMD program with a graphical user interface that allows constructing a thermodynamic and kinetic model of a given process simulated by large-scale molecular dynamics. The tool is specially designed for analyzing metadynamics based simulations. The huge amount of diverse structures generated during such a simulation is partitioned into a set of microstates (i.e. structures with similar values of the collective variables). Their relative free energies are then computed by a weighted-histogram procedure and the most relevant free energy wells are identified by diagonalization of the rate matrix followed by a commitor analysis. All this procedure leads to a convenient representation of the metastable states and long-time kinetics of the system which can be compared with experimental data. The tool allows to seamlessly switch between a collective variables space representation of microstates and their atomic structure representation, which greatly facilitates the set-up and analysis of molecular dynamics simulations. METAGUI is based on the output format of the PLUMED plugin, making it compatible with a number of different molecular dynamics packages like AMBER, NAMD, GROMACS and several others. The METAGUI source files can be downloaded from the PLUMED web site (http://www.plumed-code.org).

Optimizing the Performance of Bias-Exchange Metadynamics: Folding a 48-Residue LysM Domain Using a Coarse-Grained Model

Computer simulation of complex conformational transitions in biomolecules, such as protein folding, is considered one of the main goals of computational chemistry. A recently developed methodology, bias-exchange metadynamics, was successfully used to reversibly fold some small globular proteins. The objective of this work is to further improve this promising technique. This is accomplished by searching for the optimal set of parameters that enable folding a 48 amino acid protein, 1E0G , in the shortest possible time, using a coarse-grained force field UNRES. It is shown that bias-exchange metadynamics, if appropriately optimized, allows finding the folded state of 1E0G significantly faster than normal replica exchange.

Two-state dynamics of the SH3–SH2 tandem of Abl kinase and the allosteric role of the N-cap

Significance There is increasing interest in developing pharmacological strategies to inhibit the allosteric regulatory mechanisms of signaling enzymes. The Abl tyrosine kinase is a prominent target, due to its ubiquitous cellular role and its involvement in cancer. Here, computational and experimental methods are used in synergy to probe the mechanism of the regulatory unit of Abl, whose dual function is to inhibit the enzyme and to mediate its interaction with other signaling proteins. Our results provide insights into the thermodynamic basis for the mechanism of Abl autoinhibition and activation and expand our understanding of the principles that govern modular domain organization. The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3–SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3–SH2 connector, which involve a phosphorylation site. We also show that the SH3–SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3–SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization.

Molecular Description of the Propagation of Chirality from Molecules to Complex Systems: Different Mechanisms Controlled by Hydrophobic Interactions

In this work a combined theoretical and experimental approach was used to elucidate and describe at the molecular level the basic interactions that drive the transfer of the chiral information from chiral surfactant molecules to dye/surfactant assemblies. It was found that both hydrophobic interactions and relative concentrations strongly influence the chiroptical features of the heteroaggregates. In particular it was observed that, depending on the length of the surfactant hydrophobic chain, the chiral information is transferred to the dye by stabilizing an enantiomer either of a chiral conformer or of a chiral topological arrangement. These findings underline the role of hydrophobic interactions in the transfer of chirality and provide an example of the potential of in silico simulations for providing an accurate description of the process of chirality propagation.

Following Easy Slope Paths on a Free Energy Landscape: The Case Study of the Trp-Cage Folding Mechanism

In this work a new method for the automatic exploration and calculation of multidimensional free energy landscapes is proposed. Inspired by metadynamics, it uses several collective variables that are relevant for the investigated process and a bias potential that discourages the sampling of already visited configurations. The latter potential allows escaping a local free energy minimum following the direction of slow motions. This is different from metadynamics in which there is no specific direction of the biasing force and the computational effort increases significantly with the number of collective variables. The method is tested on the Ace-Ala3-Nme peptide, and then it is applied to investigate the Trp-cage folding mechanism. For this protein, within a few hundreds of nanoseconds, a broad range of conformations is explored, including nearly native ones, initiating the simulation from a completely unfolded conformation. Finally, several folding/unfolding trajectories give a systematic description of the Trp-cage folding pathways, leading to a unified view for the folding mechanisms of this protein. The proposed mechanism is consistent with NMR chemical shift data at increasing temperature and recent experimental observations pointing to a pivotal role of secondary structure elements in directing the folding process toward the native state.

Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins

Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na+-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581T, and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.