Sop‐GPU: Accelerating biomolecular simulations in the centisecond timescale using graphics processors

Theoretical exploration of fundamental biological processes involving the forced unraveling of multimeric proteins, the sliding motion in protein fibers and the mechanical deformation of biomolecular assemblies under physiological force loads is challenging even for distributed computing systems. Using a C_α‐based coarse‐grained self organized polymer (SOP) model, we implemented the Langevin simulations of proteins on graphics processing units (SOP‐GPU program). We assessed the computational performance of an end‐to‐end application of the program, where all the steps of the algorithm are running on a GPU, by profiling the simulation time and memory usage for a number of test systems. The ～90‐fold computational speedup on a GPU, compared with an optimized central processing unit program, enabled us to follow the dynamics in the centisecond timescale, and to obtain the force‐extension profiles using experimental pulling speeds (v_f = 1–10 μm/s) employed in atomic force microscopy and in optical tweezers‐based dynamic force spectroscopy. We found that the mechanical molecular response critically depends on the conditions of force application and that the kinetics and pathways for unfolding change drastically even upon a modest 10‐fold increase in v_f. This implies that, to resolve accurately the free energy landscape and to relate the results of single‐molecule experiments in vitro and in silico, molecular simulations should be carried out under the experimentally relevant force loads. This can be accomplished in reasonable wall‐clock time for biomolecules of size as large as 10⁵ residues using the SOP‐GPU package. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Characterization of VAMP‐2 in the lung: implication in lung surfactant secretion

Lung surfactant is crucial for reducing the surface tension of alveolar space, thus preventing the alveoli from collapse. Lung surfactant is synthesized in alveolar epithelial type II cells and stored in lamellar bodies before being released via the fusion of lamellar bodies with the apical plasma membrane. SNAREs (soluble N‐ethylmaleimide‐sensitive fusion protein‐attachment protein receptors) play an essential role in membrane fusion. We have previously demonstrated the requirement of t‐SNARE (target SNARE) proteins, syntaxin 2 and SNAP‐23 (N‐ethylmaleimide‐sensitive factor‐attachment protein 23), in regulated surfactant secretion. Here, we characterized the distribution of VAMPs (vesicle‐associated membrane proteins) in rat lung and alveolar type II cells. VAMP‐2, ?3 and ?8 are shown in type II cells at both mRNA and protein levels. VAMP‐2 and ?8 were enriched in LB (lamellar body) fraction. Immunochemistry studies indicated that VAMP‐2 was co‐localized with the LB marker protein, LB‐180. Functionally, the cytoplasmic domain of VAMP‐2, but not VAMP‐8 inhibited surfactant secretion in type II cells. We suggest that VAMP‐2 is the v‐SNARE (vesicle SNARE) involved in regulated surfactant secretion.     

Coarse‐grained simulations of proton‐dependent conformational changes in lactose permease

During lactose/H⁺ symport, the Escherichia coli lactose permease (LacY) undergoes a series of global conformational transitions between inward‐facing (open to cytoplasmic side) and outward‐facing (open to periplasmic side) states. However, the exact local interactions and molecular mechanisms dictating those large‐scale structural changes are not well understood. All‐atom molecular dynamics simulations have been performed to investigate the molecular interactions involved in conformational transitions of LacY, but the simulations can only explore early or partial global structural changes because of the computational limits (< 100 ns). In this work, we implement a hybrid force field that couples the united‐atom protein models with the coarse‐grained MARTINI water/lipid, to investigate the proton‐dependent dynamics and conformational changes of LacY. The effects of the protonation states on two key glutamate residues (Glu325 and Glu269) have been studied. Our results on the salt‐bridge dynamics agreed with all‐atom simulations at early short time period, validating our simulations. From our microsecond simulations, we were able to observe the complete transition from inward‐facing to outward‐facing conformations of LacY. Our results showed that all helices have participated during the global conformational transitions and helical movements of LacY. The inter‐helical distances measured in our simulations were consistent with the double electron‐electron resonance experiments at both cytoplasmic and periplasmic sides. Our simulations indicated that the deprotonation of Glu325 induced the opening of the periplasmics side and partial closure of the cytoplasmic side of LacY, while protonation of the Glu269 caused a stable cross‐domain salt‐bridge (Glu130‐Arg344) and completely closed the cytoplasmic side. Proteins 2016; 84:1067–1074. © 2016 Wiley Periodicals, Inc.     

Molecular modeling of the binding modes of the iron‐sulfur protein to the Jac1 co‐chaperone from Saccharomyces cerevisiae by all‐atom and coarse‐grained approaches

The iron‐sulfur protein 1 (Isu1) and the J‐type co‐chaperone Jac1 from yeast are part of a huge ATP‐dependent system, and both interact with Hsp70 chaperones. Interaction of Isu1 and Jac1 is a part of the iron‐sulfur cluster biogenesis system in mitochondria. In this study, the structure and dynamics of the yeast Isu1–Jac1 complex has been modeled. First, the complete structure of Isu1 was obtained by homology modeling using the I‐TASSER server and YASARA software and thereafter tested for stability in the all‐atom force field AMBER. Then, the known experimental structure of Jac1 was adopted to obtain initial models of the Isu1–Jac1 complex by using the ZDOCK server for global and local docking and the AutoDock software for local docking. Three most probable models were subsequently subjected to the coarse‐grained molecular dynamics simulations with the UNRES force field to obtain the final structures of the complex. In the most probable model, Isu1 binds to the left face of the Γ‐shaped Jac1 molecule by the β‐sheet section of Isu1. Residues L₁₀₅, L₁₀₉, and Y₁₆₃ of Jac1 have been assessed by mutation studies to be essential for binding (Ciesielski et al., J Mol Biol 2012; 417:1–12). These residues were also found, by UNRES/molecular dynamics simulations, to be involved in strong interactions between Isu1 and Jac1 in the complex. Moreover, N₉₅, T₉₈, P₁₀₂, H₁₁₂, V₁₅₉, L₁₆₇, and A₁₇₀ of Jac1, not yet tested experimentally, were also found to be important in binding. Proteins 2015; 83:1414–1426. © 2015 Wiley Periodicals, Inc.     

Asymmetric structure and domain binding interfaces of human tyrosyl‐tRNA synthetase studied by molecular dynamics simulations

Human tyrosyl‐tRNA synthetase (HsTyrRS) is composed of two structural modules: N‐terminal catalytic core and an EMAP II‐like C‐terminal domain. The structures of these modules are known, but no crystal structure of the full‐length HsTyrRS is currently available. An all‐atom model of the full‐length HsTyrRS was developed in this work. The structure, dynamics, and domain binding interfaces of HsTyrRS were investigated by extensive molecular dynamics (MD) simulations. Our data suggest that HsTyrRS in solution consists of a number of compact asymmetric conformations, which differ significantly by their rigidity, internal mobility, orientation of C‐terminal modules, and the strength of interdomain binding. Interfaces of domain binding obtained in MD simulations are in perfect agreement with our previous coarse‐grained hierarchical rotations technique simulations. Formation of the hydrogen bonds between R93 residue of the ELR cytokine motif and the residues A340 and E479 in the C‐module was observed. This observation supports the idea that the lack of cytokine activity in the full‐length HsTyrRS is explained by interactions between N‐modules and C‐modules, which block the ELR motif. Copyright © 2013 John Wiley & Sons, Ltd.     

Coarse-Grain Simulations Reveal Movement of the Synaptobrevin C-Terminus in Response to Piconewton Forces

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal transmembrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Application of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct application of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ～70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex.     

Folding and stability of helical bundle proteins from coarse‐grained models

We develop a coarse‐grained model where solvent is considered implicitly, electrostatics are included as short‐range interactions, and side‐chains are coarse‐grained to a single bead. The model depends on three main parameters: hydrophobic, electrostatic, and side‐chain hydrogen bond strength. The parameters are determined by considering three level of approximations and characterizing the folding for three selected proteins (training set). Nine additional proteins (containing up to 126 residues) as well as mutated versions (test set) are folded with the given parameters. In all folding simulations, the initial state is a random coil configuration. Besides the native state, some proteins fold into an additional state differing in the topology (structure of the helical bundle). We discuss the stability of the native states, and compare the dynamics of our model to all atom molecular dynamics simulations as well as some general properties on the interactions governing folding dynamics. Proteins 2013; 81:1200–1211. © 2013 Wiley Periodicals, Inc.     

Inferring a weighted elastic network from partial unfolding with coarse‐grained simulations

A number of studies have demonstrated that simple elastic network models can reproduce experimental B‐factors, providing insights into the structure–function properties of proteins. Here, we report a study on how to improve an elastic network model and explore its performance by predicting the experimental B‐factors. Elastic network models are built on the experimental coordinates, and they only take the pairs of atoms within a given cutoff distance r_c into account. These models describe the interactions by elastic springs with the same force constant. We have developed a method based on numerical simulations with a simple coarse‐grained force field, to attribute weights to these spring constants. This method considers the time that two atoms remain connected in the network during partial unfolding, establishing a means of measuring the strength of each link. We examined two different coarse‐grained force fields and explored the computation of these weights by unfolding the native structures. Proteins 2014; 82:119–129. © 2013 Wiley Periodicals, Inc.     

Role of Varp,a Rab21 exchange factor and TI‐VAMP/VAMP7 partner,in neurite growth

The vesicular soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin‐insensitive vesicle‐associated membrane protein (TI‐VAMP/VAMP7) was previously shown to mediate an exocytic pathway involved in neurite growth, but its regulation is still largely unknown. Here we show that TI‐VAMP interacts with the Vps9 domain and ankyrin‐repeat‐containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21, through a specific domain herein called the interacting domain (ID). Varp, TI‐VAMP and Rab21 co‐localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in the shaft of neurites. Silencing the expression of Varp by RNA interference or expressing ID or a form of Varp deprived of its Vps9 domain impairs neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI‐VAMP.     

Escherichia coli Exposure Inhibits Exocytic SNARE‐Mediated Membrane Fusion in Mast Cells

Mast cells orchestrate the allergic response through the release of proinflammatory mediators, which is driven by the fusion of cytoplasmic secretory granules with the plasma membrane. During this process, SNARE proteins including Syntaxin4, SNAP23 and VAMP8 play a key role. Following stimulation, the kinase IKKβ interacts with and phosphorylates the t‐SNARE SNAP23. Phosphorylated SNAP23 then associates with Syntaxin4 and the v‐SNARE VAMP8 to form a ternary SNARE complex, which drives membrane fusion and mediator release. Interestingly, mast cell degranulation is impaired following exposure to bacteria such as Escherichia coli. However, the molecular mechanism(s) by which this occurs is unknown. Here, we show that E. coli exposure rapidly and additively inhibits degranulation in the RBL‐2H3 rat mast cell line. Following co‐culture with E. coli, the interaction between IKKβ and SNAP23 is disrupted, resulting in the hypophosphorylation of SNAP23. Subsequent formation of the ternary SNARE complex between SNAP23, Syntaxin4 and VAMP8 is strongly reduced. Collectively, these results demonstrate that E. coli exposure inhibits the formation of VAMP8‐containing exocytic SNARE complexes and thus the release of VAMP8‐dependent granules by interfering with SNAP23 phosphorylation.      

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

Human age‐onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens γD‐crystallin protein and its isolated domains. A partially unfolded folding intermediate of γD‐crystallin is detected in simulations with its C‐terminal domain (C‐td) folded and N‐terminal domain (N‐td) unstructured, in excellent agreement with biochemical experiments. Our simulations strongly indicate that the stability and the folding mechanism of the N‐td are regulated by the interdomain interactions, consistent with experimental observations. A hydrophobic folding core was identified within the C‐td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non‐native surface salt‐bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non‐native Glu135‐Arg142 salt‐bridge causes significant destabilization to the folding core of the isolated C‐td, which, in turn, induces unfolding of the N‐td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of γD‐crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the γD‐crystallin surface might lead to unfolding and subsequent aggregation.     

Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development

Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N‐ethylmaleimide‐sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI‐VAMP/VAMP7 and cellubrevin/VAMP3, two v‐SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI‐VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development.     

Opposing functions of two sub‐domains of the SNARE‐complex in neurotransmission

The SNARE‐complex consisting of synaptobrevin‐2/VAMP‐2, SNAP‐25 and syntaxin‐1 is essential for evoked neurotransmission and also involved in spontaneous release. Here, we used cultured autaptic hippocampal neurons from Snap‐25 null mice rescued with mutants challenging the C‐terminal, N‐terminal and middle domains of the SNARE‐bundle to dissect out the involvement of these domains in neurotransmission. We report that the stabilities of two different sub‐domains of the SNARE‐bundle have opposing functions in setting the probability for both spontaneous and evoked neurotransmission. Destabilizing the C‐terminal end of the SNARE‐bundle abolishes spontaneous neurotransmitter release and reduces evoked release probability, indicating that the C‐terminal end promotes both modes of release. In contrast, destabilizing the middle or deleting the N‐terminal end of the SNARE‐bundle increases both spontaneous and evoked release probabilities. In both cases, spontaneous release was affected more than evoked neurotransmission. In addition, the N‐terminal deletion delays vesicle priming after a high‐frequency train. We propose that the stability of N‐terminal two‐thirds of the SNARE‐bundle has a function for vesicle priming and limiting spontaneous release.     

Microsecond simulations of the folding/unfolding thermodynamics of the Trp‐cage miniprotein

We study the unbiased folding/unfolding thermodynamics of the Trp‐cage miniprotein using detailed molecular dynamics simulations of an all‐atom model of the protein in explicit solvent using the Amberff99SB force field. Replica‐exchange molecular dynamics simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states at two densities. The obtained ensembles are shown to reach equilibrium in the 1 μs/replica timescale. The total simulation time used in the calculations exceeds 100 μs. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, ΔG(P, T), over the whole region of temperatures and pressures sampled in the simulations. The ΔG(P, T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure‐induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (T_f = 321 K), free energy, and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semiempirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Coarse‐ and fine‐grained models for proteins: Evaluation by decoy discrimination

Coarse‐grained models for protein structure are increasingly used in simulations and structural bioinformatics. In this study, we evaluated the effectiveness of three granularities of protein representation based on their ability to discriminate between correctly folded native structures and incorrectly folded decoy structures. The three levels of representation used one bead per amino acid (coarse), two beads per amino acid (medium), and all atoms (fine). Multiple structure features were compared at each representation level including two‐body interactions, three‐body interactions, solvent exposure, contact numbers, and angle bending. In most cases, the all‐atom level was most successful at discriminating decoys, but the two‐bead level provided a good compromise between the number of model parameters which must be estimated and the accuracy achieved. The most effective feature type appeared to be two‐body interactions. Considering three‐body interactions increased accuracy only marginally when all atoms were used and not at all in medium and coarse representations. Though two‐body interactions were most effective for the coarse representations, the accuracy loss for using only solvent exposure or contact number was proportionally less at these levels than in the all‐atom representation. We propose an optimization method capable of selecting bead types of different granularities to create a mixed representation of the protein. We illustrate its behavior on decoy discrimination and discuss implications for data‐driven protein model selection. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

An activated Q‐SNARE/SM protein complex as a possible intermediate in SNARE assembly

Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18‐1 as a possible acceptor complex for the R‐SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18‐1 remains tethered by the N‐terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1‐65) was able to bind to the syntaxin1:SNAP25:Munc18‐1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R‐SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF. Based on these findings, we consider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly.