Targeted Molecular Dynamics Simulations Research Articles

DESP: Deep Enhanced Sampling of Proteins' Conformation Spaces Using AI-Inspired Biasing Forces.

The molecular structures (i.e., conformation spaces, CS) of bio-macromolecules and the dynamics that molecules exhibit are crucial to the understanding of the basis of many diseases and in the continuous attempts to retarget known drugs/medications, improve the efficacy of existing drugs, or develop novel drugs. These make a better understanding and the exploration of the CS of molecules a research hotspot. While it is generally easy to computationally explore the CS of small molecules (such as peptides and ligands), the exploration of the CS of a larger biomolecule beyond the local energy well and beyond the initial equilibrium structure of the molecule is generally nontrivial and can often be computationally prohibitive for molecules of considerable size. Therefore, research efforts in this area focus on the development of ways that systematically favor the sampling of new conformations while penalizing the resampling of previously sampled conformations. In this work, we present Deep Enhanced Sampling of Proteins’ Conformation Spaces Using AI-Inspired Biasing Forces (DESP), a technique for enhanced sampling that combines molecular dynamics (MD) simulations and deep neural networks (DNNs), in which biasing potentials for guiding the MD simulations are derived from the KL divergence between the DNN-learned latent space vectors of [a] the most recently sampled conformation and those of [b] the previously sampled conformations. Overall, DESP efficiently samples wide CS and outperforms conventional MD simulations as well as accelerated MD simulations. We acknowledge that this is an actively evolving research area, and we continue to further develop the techniques presented here and their derivatives tailored at achieving DNN-enhanced steered MD simulations and DNN-enhanced targeted MD simulations.

Beryllium-driven structural evolution at the divertor surface

Erosion of the beryllium first wall material in tokamak reactors has been shown to result in transport and deposition on the tungsten divertor. Experimental studies of beryllium implantation in tungsten indicate that mixed W–Be intermetallic deposits can form, which have lower melting temperatures than tungsten and can trap tritium at higher rates. To better understand the formation and growth rate of these intermetallics, cumulative molecular dynamics (MD) simulations of both high and low energy beryllium deposition in tungsten were performed. In both cases, a W–Be mixed material layer (MML) emerged at the surface within several nanoseconds, either through energetic implantation or a thermally-activated exchange mechanism, respectively. While some ordering of the material into intermetallics occurred, fully ordered structures did not emerge from the deposition simulations. Targeted MD simulations of the MML to further study the rate of Be diffusion and intermetallic growth rates indicate that for both cases, the gradual re-structuring of the material into an ordered intermetallic layer is beyond accessible MD time scales(⩽1 μs). However, the rapid formation of the MML within nanoseconds indicates that beryllium deposition can influence other plasma species interactions at the surface and begin to alter the tungsten material properties. Therefore, beryllium deposition on the divertor surface, even in small amounts, is likely to cause significant changes in plasma-surface interactions and will need to be considered in future studies.

Prediction of Absolute Binding and Unbinding Kinetics on Timescales of a Minute from Sub-Microsecond MD Simulations

The prediction of absolute rates of protein-ligand complex association and dissociation that have an impact on pharmacological efficacy is problematic, as the intrinsic timescales of seconds to hours are far outside the scope of current molecular dynamics (MD) techniques. We here present a novel approach to coarse-grain fully atomistic dynamics based on principles from nonequilibrium statistical mechanics to gain access to such timescales: using dissipation-corrected targeted MD simulations, we enforce ligand unbinding and use the resulting bias force to calculate free energies and friction profiles along resulting unbinding paths.

Binding of HasA by its transmembrane receptor HasR follows a conformational funnel mechanism

HasR in the outer membrane of Serratia marcescens binds secreted, heme-loaded HasA and translocates the heme to the periplasm to satisfy the cell’s demand for iron. The previously published crystal structure of the wild-type complex showed HasA in a very specific binding arrangement with HasR, apt to relax the grasp on the heme and assure its directed transfer to the HasR-binding site. Here, we present a new crystal structure of the heme-loaded HasA arranged with a mutant of HasR, called double mutant (DM) in the following that seemed to mimic a precursor stage of the abovementioned final arrangement before heme transfer. To test this, we performed first molecular dynamics (MD) simulations starting at the crystal structure of the complex of HasA with the DM mutant and then targeted MD simulations of the entire binding process beginning with heme-loaded HasA in solution. When the simulation starts with the former complex, the two proteins in most simulations do not dissociate. When the mutations are reverted to the wild-type sequence, dissociation and development toward the wild-type complex occur in most simulations. This indicates that the mutations create or enhance a local energy minimum. In the targeted MD simulations, the first protein contacts depend upon the chosen starting position of HasA in solution. Subsequently, heme-loaded HasA slides on the external surface of HasR on paths that converge toward the specific arrangement apt for heme transfer. The targeted simulations end when HasR starts to relax the grasp on the heme, the subsequent events being in a time regime inaccessible to the available computing power. Interestingly, none of the ten independent simulation paths visits exactly the arrangement of HasA with HasR seen in the crystal structure of the mutant. Two factors which do not exclude each other could explain these observations: the double mutation creates a non-physiologic potential energy minimum between the two proteins and /or the target potential in the simulation pushes the system along paths deviating from the low-energy paths of the native binding processes. Our results support the former view, but do not exclude the latter possibility.

Molecular Energetics of Doxorubicin Pumping by Human P-Glycoprotein.

The pumping of antitumor drugs by P-glycoprotein (P-gp) causes multidrug resistance (MDR) and consequent failure of chemotherapy. However, the understanding on the molecular mechanism of P-gp for transporting substrates is still far from adequate. Herein, the transport of a typical antitumor drug, doxorubicin, by P-gp is investigated using targeted molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis. The MM-PBSA analysis identifies the driving forces for the transport of doxorubicin toward the extracellular space as electrostatic repulsions in the initial stage, which are contributed by positively charged residues (R148, K181, K189, K285, K291, K734, R789, K826, K934, and K1000) and then hydrophobic interactions provided by hydrophobic residues (L65, M69, F336, I340, F343, Y953, V982, F983, and M986). The contributions of these residues are further validated by targeted MD simulations, which shows blocked pumping after the mutation of these important residues to glycine. The MM-PBSA and minimum distance analyses of each residue during the transport reveal that the positively charged residues promote the transport of doxorubicin through long-range electrostatic repulsions and the hydrophobic residues provide a pathway through continuous hydrophobic interactions to maintain the transport. The results have thus provided molecular insights into the function of P-gp and would be beneficial in the design of potent P-gp inhibitors against MDR in the medication of cancers.

Chemo-Mechanical Coupling in the Transport Cycle of a Heme ABC Transporter

The heme importer from pathogenic bacteria is a member of the ATP-binding cassette (ABC) transporter family, which uses the energy of ATP-binding and hydrolysis for extensive conformational changes. Previous studies have indicated that conformational changes after heme translocation are triggered by ATP-binding to nucleotide binding domains (NBDs) and then, in turn, induce conformational transitions of the transmembrane domains (TMDs). In this study, we applied a template-based iterative all-atom molecular dynamics (MD) simulation to predict the ATP-bound outward-facing conformation of the Burkholderia cenocepacia heme importer BhuUV-T. The resulting model showed a stable conformation of the TMD with the cytoplasmic gate in the closed state and the periplasmic gate in the open state. Furthermore, targeted MD simulation predicted the intermediate structure of an occluded form (Occ) with bound ATP, in which both ends of the heme translocation channel are closed. The MD simulation of the predicted Occ revealed that Ser147 on the ABC signature motifs (LSGG[Q/E]) of NBDs occasionally flips and loses the active conformation required for ATP-hydrolysis. The flipping motion was found to be coupled to the inter-NBD distance. Our results highlight the functional significance of the signature motif of ABC transporters in regulation of ATPase and chemo-mechanical coupling mechanism.

Principal component analysis of nonequilibrium molecular dynamics simulations.

Principal component analysis (PCA) represents a standard approach to identify collective variables {xi} = x, which can be used to construct the free energy landscape ΔG(x) of a molecular system. While PCA is routinely applied to equilibrium molecular dynamics (MD) simulations, it is less obvious as to how to extend the approach to nonequilibrium simulation techniques. This includes, e.g., the definition of the statistical averages employed in PCA as well as the relation between the equilibrium free energy landscape ΔG(x) and the energy landscapes ΔG(x) obtained from nonequilibrium MD. As an example for a nonequilibrium method, "targeted MD" is considered which employs a moving distance constraint to enforce rare transitions along some biasing coordinate s. The introduced bias can be described by a weighting function P(s), which provides a direct relation between equilibrium and nonequilibrium data, and thus establishes a well-defined way to perform PCA on nonequilibrium data. While the resulting distribution P(x) and energy ΔG∝lnP will not reflect the equilibrium state of the system, the nonequilibrium energy landscape ΔG(x) may directly reveal the molecular reaction mechanism. Applied to targeted MD simulations of the unfolding of decaalanine, for example, a PCA performed on backbone dihedral angles is shown to discriminate several unfolding pathways. Although the formulation is in principle exact, its practical use depends critically on the choice of the biasing coordinate s, which should account for a naturally occurring motion between two well-defined end-states of the system.

Structural Basis for Dengue Virus Antibody Dependent Maturation

Event Abstract Back to Event Structural basis for dengue virus antibody dependent maturation Jan K. Marzinek1 and Peter J. Bond1* 1 Bioinformatics Institute (A*STAR), Singapore Background Dengue virus (DENV) is responsible for millions of infections a year around the world. DENV is composed of an RNA genome complexed with capsid (C) proteins, which are surrounded by envelope (E) and membrane (M) proteins embedded within a lipid bilayer. In nascent viral particles, the pre-membrane (prM) protein caps the fusion loop of the E protein, making it non-fusogenic. During maturation, cleavage of prM into the M protein and release of pr leads to a conformational change in the E protein that makes it fusion-competent. Interestingly, DENV is known to be released from cells under different states of maturation. Fully immature DENV (immDENV) is non-infectious. However, its infectivity can be enhanced when DENV is targeted by host anti-prM antibodies. The molecular basis for this antibody-dependent enhancement has until recently been unknown. Methods In this work, cryo-electron microscopy (cryo-EM) structures of the immDENV:anti-prM complex at acidic and neutral pH conditions mimicking the endosomal and extracellular environments respectively were analyzed via integrative modeling and simulation approaches. Results At acidic pH, an intermediate structure of the DENV maturation pathway has been solved with fewer antibodies bound in comparison to neutral pH. Based on previously developed multiscale models of the DENV particle, we employed sets of targeted molecular dynamics (TMD) simulations in order to trigger the maturation transition at low pH. Hydrogen-deuterium exchange mass spectroscopy and Elisa measurements confirmed that the affinity of the antibody:pr complexes are stronger than those of the pr:E complexes, and the TMD simulations revealed dislodgement of antibody:pr from the E protein surface during the conformational transition due to steric clashes. Conclusion Here, we provide the detailed molecular mechanism by which anti-prM antibodies enhance maturation of DENV in the acidic endosomal environment, leading to exposure of the E protein fusion loops for subsequent endosomal membrane binding and fusion. Acknowledgements National Research Foundation: CRP Grant Number: NRF2017-CRP001-027 Keywords: Dengue virus maturation, cryo-electron microscopy, antibody, PRM, Molecular dynamic simulations Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Oral Presentation Topic: Infectious diseases Citation: Marzinek JK and Bond PJ (2019). Structural basis for dengue virus antibody dependent maturation. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00082 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Peter J Bond, Bioinformatics Institute (A*STAR), Singapore, Singapore, peterjb@bii.a-star.edu.sg Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jan K Marzinek Peter J Bond Google Jan K Marzinek Peter J Bond Google Scholar Jan K Marzinek Peter J Bond PubMed Jan K Marzinek Peter J Bond Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Identification and Validation of Reaction Coordinates Describing Protein Functional Motion: Hierarchical Dynamics of T4 Lysozyme.

While adequately chosen reaction coordinates are expected to reveal the mechanism of a dynamical process, it proves to be notoriously difficult to model the complex structural rearrangements of a macromolecule by a low-dimensional collective coordinate. Adopting the hinge-bending motion of T4 lysozyme (T4L) as a prominent example and performing a 50 μs long unbiased molecular dynamics (MD) simulation of T4L, a general strategy to identify reaction coordinates of protein functional dynamics is developed. As a systematic method to reduce the dimensionality of the dynamics, first various types of principal component analyses are employed, and it is shown that the applicability and outcome of the approach crucially depends on the type of input coordinates used. In a second step, prospective candidates for a reaction coordinate are tested by studying the molecule's response to external pulling along the coordinate, using targeted MD simulations. While trying to directly enforce the open-closed transition does not recover the two-state behavior of T4L, this transition is triggered by a locking mechanism, by which the side chain of Phe4 changes from a solvent-exposed to a hydrophobically buried state. The mechanism is found to stabilize the open and closed states of T4L and thereby causes their relatively long lifetime of ∼10 μs. In extension of the usual two-state picture, a four-state model of the functional motion of T4L is proposed, which describes a hierarchical coupling of the fast nanosecond opening-closing motion and the slow microsecond locking transition.

Loop-driven conformational transition between the alternative and collapsed form of prethrombin-2: targeted molecular dynamics study

Two distinct crystal structures of prethrombin-2, the alternative and collapsed forms, are elucidated by X-ray crystallogrphy. We analyzed the conformational transition from the alternative to the collapsed form employing targeted molecular dynamics (TMD) simulation. Despite small RMSD difference in the two X-ray crystal structures, some hydrophobic residues (W60d, W148, W215, and F227) show a significant difference between the two conformations. TMD simulation shows that the four hydrophobic residues undergo concerted movement from dimer to trimer transition via tetramer state in the conformational change from the alternative to the collapsed form. We reveal that the concerted movement of the four hydrophobic residues is controlled by movement of specific loop regions behind. In this paper, we propose a sequential scenario for the conformational transition from the alternative form to the collapsed form, which is partially supported by the mutant W148A simulation.

Study on the Application of the Combination of TMD Simulation and Umbrella Sampling in PMF Calculation for Molecular Conformational Transitions.

Free energy calculations of the potential of mean force (PMF) based on the combination of targeted molecular dynamics (TMD) simulations and umbrella samplings as a function of physical coordinates have been applied to explore the detailed pathways and the corresponding free energy profiles for the conformational transition processes of the butane molecule and the 35-residue villin headpiece subdomain (HP35). The accurate PMF profiles for describing the dihedral rotation of butane under both coordinates of dihedral rotation and root mean square deviation (RMSD) variation were obtained based on the different umbrella samplings from the same TMD simulations. The initial structures for the umbrella samplings can be conveniently selected from the TMD trajectories. For the application of this computational method in the unfolding process of the HP35 protein, the PMF calculation along with the coordinate of the radius of gyration (Rg) presents the gradual increase of free energies by about 1 kcal/mol with the energy fluctuations. The feature of conformational transition for the unfolding process of the HP35 protein shows that the spherical structure extends and the middle α-helix unfolds firstly, followed by the unfolding of other α-helices. The computational method for the PMF calculations based on the combination of TMD simulations and umbrella samplings provided a valuable strategy in investigating detailed conformational transition pathways for other allosteric processes.

Understanding the conformation transition in the activation pathway of β2 adrenergic receptor via a targeted molecular dynamics simulation.

G protein coupled receptors (GPCRs) play a crucial role in regulating signal recognition and transduction through their activation. The conformation transition in the activation pathway is of particular importance for their function. However, it has been poorly elucidated due to experimental difficulties in determining the conformations and the time limitation of conventional molecular dynamics (CMD) simulation. Thus, in this work, we employed a targeted molecular dynamic (TMD) simulation to study the activation process from an inactive structure to a fully active one for β2 adrenergic receptor (β2AR). As a reference, 110 ns CMD simulations on wild β2AR and its D130N mutant were also carried out. TMD results show that there is at least an intermediate conformation cluster in the activation process, evidenced by the principal component analysis and the structural and dynamic differences of some important motifs. It is noteworthy that the activation of the ligand binding site lags the G-protein binding site, displaying uncoupled correlation. Comparisons between the CMD and TMD results show that the D130N mutation significantly speeds up ICL2 and key ionic lock to enter into the intermediate state, which to some extent facilitates the activation involved in the NPxxY, DRY region and the separation between TM3 and TM6. However, the contribution from the D130N mutation to the activation of the ligand binding site could not be observed within the scale of 110 ns time. These observations could provide novel insights into previous studies for better understanding of the activation mechanism for β2AR.

NMDA Receptor Transmembrane Domain: Structure and Mechanism of Ion Selectivity

NMDA receptors (NMDARs) are iGluR subfamilies that are activated during synaptic transmission. The voltage dependence of NMDARs differentiates them from other iGluRs: at typical neuronal resting voltages, NMDAR channels are mostly blocked by Mg2+, but when membrane voltage is depolarized, Mg2+ block is relieved, resulting in Ca2+ influx through NMDAR at postsynaptic sites. The mechanisms, by which NMDARs select Ca2+ for permeation over all other physiological ions, while binding Mg2+ and restricting its permeation, are not well understood.Recently, partially resolved medium-resolution structures of an NMDAR tetramer were published. The structures tremendously improve our knowledge and understanding of the architecture and design of the NMDARs. Yet, one of the key structural features, namely the ion selectivity filter and parts of the pore region of the ion channel itself, are missing from these structures. This region of the protein has not been resolved also in the earlier high-resolution structure of an AMPAR, a closely related iGluR family member.Previously a successful homology model of the NMDAR TMD using one of the potassium channel family members, NaK channel as a template, was shown to have good agreements with available experimental data [Siegler et. al., Nat Neurosci 15: 406–13]. Given recently released NMDAR structures and our previously developed homology model, we are now in position to propose a high resolution NMDAR TMD model that is based on a hybrid structure. We performed extensive molecular dynamics (MD) and targeted MD simulations of our NMDAR TMD domain model in lipid bilayer and water. We demonstrate that our proposed structure is stable in simulations and has a well-formed binding site for Mg2+ and Ca2+.

Frequency Response of a Protein to Local Conformational Perturbations

Signals created by local perturbations are known to propagate long distances through proteins via backbone connectivity and nonbonded interactions. In the current study, signal propagation from the flexible ligand binding loop to the rest of Protein Tyrosine Phosphatase 1B (PTP1B) was investigated using frequency response techniques. Using restrained Targeted Molecular Dynamics (TMD) potential on WPD and R loops, PTP1B was driven between its crystal structure conformations at different frequencies. Propagation of the local perturbation signal was manifested via peaks at the fundamental frequency and upper harmonics of 1/f distributed spectral density of atomic variables, such as Cα atoms, dihedral angles, or polar interaction distances. Frequency of perturbation was adjusted high enough (simulation length >∼10×period of a perturbation cycle) not to be clouded by random diffusional fluctuations, and low enough (<∼0.8 ns−1) not to attenuate the propagating signal and enhance the contribution of the side-chains to the dissipation of the signals. Employing Discrete Fourier Transform (DFT) to TMD simulation trajectories of 16 cycles of conformational transitions at periods of 1.2 to 5 ns yielded Cα displacements consistent with those obtained from crystal structures. Identification of the perturbed atomic variables by statistical t-tests on log-log scale spectral densities revealed the extent of signal propagation in PTP1B, while phase angles of the filtered trajectories at the fundamental frequency were used to cluster collectively fluctuating elements. Hydrophobic interactions were found to have a higher contribution to signal transduction between side-chains compared to the role of polar interactions. Most of in-phase fluctuating residues on the signaling pathway were found to have high identity among PTP domains, and located over a wide region of PTP1B including the allosteric site. Due to its simplicity and efficiency, the suggested technique may find wide applications in identification of signaling pathways of different proteins.

A Computational Study of the Glycine-Rich Loop of Mitochondrial Processing Peptidase

An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the α-subunit of the yeast mitochondrial processing peptidase (MPP) and its importance for the tertiary and quaternary conformation of MPP. Wild-type and GRL-deleted MPP structures were studied using non-restrained MD simulations, both in the presence and the absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at least during the first third of the substrate translocation trajectory. Further, we show that the substrate remains in contact with the GRL during the whole first half of the translocation trajectory and that hydrophobic interactions play a major role. Finally, we conclude that the GRL acts as a precisely balanced structural element, holding the MPP subunits in a partially closed conformation regardless the presence or absence of a substrate in the active site.

Conformational Transition Pathway in the Activation Process of Allosteric Glucokinase

Glucokinase (GK) is a glycolytic enzyme that plays an important role in regulating blood glucose level, thus acting as a potentially attractive target for drug discovery in the treatment of diabetes of the young type 2 and persistent hyperinsulinemic hypoglycemia of infancy. To characterize the activation mechanism of GK from the super-open state (inactive state) to the closed state (active state), a series of conventional molecular dynamics (MD) and targeted MD (TMD) simulations were performed on this enzyme. Conventional MD simulation showed a specific conformational ensemble of GK when the enzyme is inactive. Seven TMD simulations depicted a reliably conformational transition pathway of GK from the inactive state to the active state, and the components important to the conformational change of GK were identified by analyzing the detailed structures of the TMD trajectories. In combination with the inactivation process, our findings showed that the whole conformational pathway for the activation-inactivation-activation of GK is a one-direction circulation, and the active state is less stable than the inactive state in the circulation. Additionally, glucose was demonstrated to gradually modulate its binding pose with the help of residues in the large domain and connecting region of GK during the activation process. Furthermore, the obtained energy barriers were used to explain the preexisting equilibrium and the slow binding kinetic process of the substrate by GK. The simulated results are in accordance with the recent findings from the mutagenesis experiments and kinetic analyses. Our observations reveal a complicated conformational process in the allosteric protein, resulting in new knowledge about the delicate mechanisms for allosteric biological macromolecules that will be useful in drug design for targeting allosteric proteins.

Structural Transition between the Inward- and Outward-Facing States of the Glutamate Transporter in Different Lipid Environments

Excitatory amino acid transporters (EAATs) are membrane proteins responsible for the reuptake of glutamate from the synaptic cleft in the central nervous system. Crystallographic data of a bacterial EAAT homologue, GltPh, have provided structural information for this trimeric secondary active transporter in different states. Nevertheless, in contrast to other membrane proteins, such as GPCRs, little is known about the structural or functional coupling between EAATs and their membrane environment. In this context, we investigated the effect of lipid environment on the structure and dynamics of GltPh, using all-atom molecular dynamics (MD) simulations of both the outward- and inward-facing conformations of GltPh in either POPC or POPE lipid membranes. The transition between the two states was explored with a variant of targeted MD simulations (sTMD) combining stepwise targeted motion and equilibration. Both the end conformations and the transition pathways were found to be robust to the choice of lipid bilayer type (pairwise trajectory RMSDs<2.5A), implying constant contributions from the energetics of membrane deformation and hydrophobic mismatch computed with 3D-CTMD. The transition pathways connecting the end states agree substantially with the structural intermediates of a path identified recently using the method of motion planning (MP) coupled with MD (minimum RMSDs<3A). Importantly, the agreement includes the prediction that the transport-trimerization domain interface changes continuously during the transition, exhibiting in the middle a significantly reduced contact area and significantly increased solvent-accessibility compared to the end states. This remarkable consistency between simple MP and the new sTMD modeling approaches indicates that the computationally economical MP calculations provide a useful initial modeling tool for major conformational transitions in membrane proteins.

Characterizing Transition Pathways in the Transport Cycle of ABC Transporter MsbA

MsbA is a member of the ABC (ATP-binding cassette) family transporters that uses energy from ATP hydrolysis to transport various substrates across cellular membrane. The transport cycle of MsbA has been described by a simple “Alternating Access” mechanism in which the transporter changes its conformation between an outward-facing (OF) and an inward-facing (IF) state while coupling the translocation of substrates. Here, to investigate the detailed transport cycle of MsbA at atomistic resolution, we have performed molecular dynamics (MD) simulations using a combination of several computational methods. Starting from the recently solved X-ray crystal structure of MsbA in OF state, we first generate high resolution all-atom models for the two IF states (IF open and IF closed) by using targeted MD simulations. We then define two collective variables to describe the relative motion of the two transmembrane domains (TMDs) and nucleotide binding domains (NBDs), respectively. Steered MD simulations along the collective variables have been performed to induce the conformational changes between the three states (OF, IF open and IF closed) of MsbA. Assessing the energetics associated with the induced transitions, approximated by calculating the non-equilibrium work involved in going from one state to another suggest that the OF-to-IF conformational transition follows two steps: the two TMDs close first, and then the two NBDs open, as opposed to the pathway observed from initial targeted MD simulations. Taken together, these results not only provide a better understanding of the functionality of ABC transporters, but also help define a general mechanism for membrane transport process.

MurD ligase from Escherichia coli: C-terminal domain closing motion

MurD (UDP- N-acetylmuramoyl- l-alanine: d-glutamate ligase), a three domain bacterial protein, catalyzes a highly specific incorporation of the d-glutamate into the cytoplasmic intermediate UDP- N-acetyl-muramoyl- l-alanine (UMA) utilizing ATP hydrolysis to ADP and P i. This reaction is part of the biosynthetic pathway enabling bacterial peptidoglycan synthesis. During our previous investigations of this complex dynamic antibacterial target, Targeted Molecular Dynamics (TMDs) simulations were performed (Perdih et al., Proteins: Struct. Funct. Bioinf., 2007) in order to examine the UMA and ATP substrates binding process and gain insights into the structural changes that occur during the conformational transitions of the C-terminal domain from the experimentally determined open structures to its active closed conformation. Off-path simulation (OPS) technique, an extension of the well-established Replica Path Method (RPATh), was applied to compare the relative energy of the two by C-terminal domain closing motions generated by TMD simulations. The first C-terminal domain closing process commenced from the experimental open structure in which this domain is located out-of plane to the N-terminal and central domains (open structure pdb:1EEH). In the second trajectory the conformational movement is confined to this plane only (open structure pdb:1E0D). The obtained results indicated that the C-terminal domain movement occurring from the out-of-plane starting structure is coupled with much higher energy changes compared to the motion of this domain within this plane which is more freely available to the enzyme. The results nicely corroborate experimental findings from recent structural studies. The study offers valuable information for novel structure-based virtual screening campaigns exploiting protein conformational flexibility.

Computer simulations of water flux and salt permeability of the reverse osmosis FT-30 aromatic polyamide membrane

The relative permeability of salt to water across an atomistic model of the FT-30 reverse osmosis (RO) membrane is studied using molecular dynamics (MD) simulations. The membrane model is built using a heuristic approach and gives a membrane density, water solubility and flux that are in good accord with the experimental values. The salt permeability is calculated from inhomogeneous solubility–diffusion theory using ion pathways from non-equilibrium targeted MD simulations, yielding an estimated salt rejection of 99.9% that is similar to the experimental value. The encouraging agreement with experimental data of FT-30 membrane suggests that MD simulations based on atomic models offer a useful way to support the experimental exploration of RO membrane development.