Multi-microsecond Simulations Research Articles

Simulations of the Upper Critical Solution Temperature Behavior of Poly(ornithine-co-citrulline)s Using MARTINI-Based Coarse-Grained Force Fields.

Poly(ornithine-co-citrulline)s are ureido-based polymers, which were shown to exhibit tunable upper critical solution temperature (UCST) behavior, a property that can be exploited to develop thermoresponsive nanoparticles for controlled drug delivery systems. To gain insight into the driving forces that govern the formation and dissolution processes of poly(ornithine-co-citrulline) nanoparticles, a molecular dynamics (MD) simulation study has been carried out using MARTINI-based protein coarse-grained models. Multi-microsecond simulations at temperatures ranging from 280 to 370 K show that the fully reparametrized version 3.0 of MARTINI force field is able to capture the dependence on temperature of poly(ornithine-co-citrulline) aggregation and dissolution, while version 2.2 could not account for it. Furthermore, the phase separation observed in these simulations allowed us to extrapolate a phase diagram based on the Flory-Huggins theory of polymer solution, which could help in future rational design of drug delivery nanoparticles based on poly(amino acid)s.

Energetics of Substrate Binding and Conformational Change of the Bile Acid Transporter ASBTNm

The apical sodium-dependant bile acid transporter (ASBT) utilises the sodium gradient to drive the re-absorption of bile acids from the intestine, operating through an alternating access mechanism. It is of interest as a potential target for the treatment of hypercholesterolaemia as well as for drug delivery. Several structures of bacterial homologues are available, including that from Neisseria meningitidis (ASBTNM) in an inward-facing conformation with sodium and the bile acid taurocholate bound. However, less is known about the dynamic details of the ASBT transport cycle, including the nature of the conformational transition and the interplay of the substrates. We studied the behaviour of ASBTNM on an atomistic scale with molecular dynamics (MD) simulations. Multi-microsecond simulations were used to investigate the stable inward- and outward-facing states and substrate binding sites. The energetics of the conformational transition and of sodium and taurocholate binding to ASBTNM, and how this is altered in different substrate-bound configurations, were calculated using a two-stage enhanced sampling approach, in which semi-quantitative energetic landscapes were first obtained from short metadynamics simulations and then used to guide parameter selection for more accurate Bias Exchange Umbrella Sampling (BEUS) simulations. Together, these results represent an important step towards understanding the complete transport cycle of ASBT.

The Lipids that Get the Invite to a Caveolar Neck Party

Caveolae are plasma membrane (PM) invaginations that appear as bulbs protruding into the cytoplasm. individual bulbs are connected to the bulk membrane by a highly curved neck with an internal pore. Like other highly curved biological structures (e.g., pores created by membrane compartment fusion and clathrin coated endocytic sites before the vesicle pinches off), caveolar necks are created by cellular protein machinery, and the work done by proteins to create and sustain the high curvature depends on the lipid membrane material properties and distributions. Understanding and quantifying lipid equilibrium distributions requires atomistic detail to resolve specific interactions, but also long timescales to allow sufficient lipid diffusion. To meet both of these demands, we used the special-use Anton supercomputer to attain continuous, long timescale trajectories of lipid diffusion on two pores with different internal radii and a planar bilayer as a control. We used a PM mimetic that balances the necessary lipidic complexity with the necessary simplicity to quantify important lipid-lipid interaction motifs and redistributions on a timescale accessible to all-atom simulations. The multi-microsecond simulations allowed us to test two core hypotheses: i) that ganglioside GM3 and palmitoyl sphingomyelin redistribute to areas of positive curvature, and ii) that cholesterol will preferentially flip-flop into the outer leaflet. Because of the diverse curvature accessible on a caveolar pore, the simulations also allow insight into whether lipids redistribute with respect to both net curvature and Gaussian curvature. Preliminary results indicate that cholesterol is depleted in the narrower neck pore and that phosphatidylcholine lipids are enriched in pore neck pores.

Capturing Substrate Translocation in an ABC Exporter at the Atomic Level.

ATP-binding cassette (ABC) transporters chemomechanically couple ATP binding and hydrolysis to large-scale conformational changes, ultimately leading to substrate translocation across biological membranes. Despite recent progress in the structure determination of substrate-bound ABC exporters, the inherently dynamic mechanism of substrate transport remains unclear at the atomic level. In this work, we capture substrate translocation in the heterodimeric ABC exporter TM287/288 from the hyperthermophilic bacterium Thermotoga maritima using all-atom molecular dynamics (MD) simulations. Unguided multimicrosecond simulations at 375 K show how the drugs daunorubicin and verapamil, which were initially docked into the ABC transporter, get translocated through the exporter by following its large-scale alternating access conformational transitions between an inward-facing (IF) and an outward-facing (OF) conformation. Triggered by the affinity difference due to differential solvation of the binding cavity in the IF and OF conformations, the substrates unbind from the OF transporter and partition into the lipid bilayer. While daunorubicin is stably inserted into the outer leaflet of the bilayer, verapamil dynamically flip flops between the bilayer leaflets, possibly rendering its net transport futile.

What Makes a Good Pore Former: A Study of Synthetic Melittin Derivatives

Pore formation by membrane-active peptides, naturally encountered in innate immunity and infection, could have important medical and technological applications. Recently, the well-studied lytic peptide melittin has formed the basis for the development of combinatorial libraries from which potent pore-forming peptides have been derived, optimized to work under different conditions. We investigate three such peptides, macrolittin70, which is most active at neutral pH; pHD15, which is active only at low pH; and MelP5_Δ6, which was rationally designed to be active at low pH but formed only small pores. There are three, six, and six acidic residues in macrolittin70, pHD15, and MelP5_Δ6, respectively. We perform multi-microsecond simulations in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) of hexamers of these peptides starting from transmembrane orientations at neutral pH (all residues at standard protonation), low pH (acidic residues and His protonated), and highly acidic environments in which C-termini are also protonated. Previous simulations of the parent peptides melittin and MelP5 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are repeated in POPC. We find that the most potent pore-forming peptides exhibit strong interpeptide interactions, including salt bridges, H-bonds, and polar interactions. Protonation of the C-terminus promotes helicity and pore size. The proximity of the peptides allows fewer lipid headgroups to line the pores than in previous simulations, making the pores intermediate between barrel stave and toroidal. Based on these structures and geometrical arguments, we attempt to rationalize the factors that under different conditions can increase or decrease pore stability and propose mutations that could be tested experimentally.

Allosteric Network Analysis in the NMDA Receptor

The NMDA receptor (NMDAR) is a ligand-gated ion channel binding glutamate and glycine, it is present in postsynaptic neurons. It is part of the glutamate receptor family and plays an essential function in synaptic plasticity and memory formation. Dysregulation of this receptor has been implicated in a range of conditions including Alzheimer's, epilepsy, schizophrenia and depression. The NMDAR is therefore of great therapeutic interest, and modulation of the receptor has shown promising results. One method used to gain additional insight into the intricacies of the NMDAR is molecular dynamics. This can provide atomistic scale insight into the interactions between protein and ligands, as well as any alterations in protein dynamics based on ligand binding, something that is of great interest in drug development. A significant challenge in simulations of the NMDAR is the size of the protein, as well as the number of loosely connected domains. Crystal structures have resolved the structure of the amino-terminal, ligand-binding and trans-membrane domains, together making up over 3000 residues, leading to simulation systems over 500,000 atoms in size, resulting in sampling issues. The large size of the system makes these simulations expensive to run and the number of domains means long timescales are required to ensure inter-domain communication is achieved and the protein reaches an equilibrated state. For these reasons alternative ways of gaining atomistic insight than expensive multi-microsecond simulations are required. In this work we used allosteric network analysis to compare the inter-domain communication pathways in the presence and absence of ligands, showing clear differences between the apo state and the glutamate and glycine bound state. This could be of great interest in small molecule research where demonstrating the ability of molecules to distrupt "normal" communication pathways could help guide drug development.

Molecular Structure of the Long Periodicity Phase in the Stratum Corneum.

All-atom models of the long periodicity phase (LPP) in the stratum corneum (SC) are studied using bilayer-slab-bilayer (sandwich) structures and multi-microsecond simulations. Linoleate promotes melting of the interior slab, which contains ceramide (Cer) in the posturing chain conformation, a structurally distinct conformation from full chain extension. The mechanism of Cer transitioning into full extension is characterized by initial anchoring of the head-proximal carbons and occurs over tens of nanoseconds. Free fatty acids translocate through the interior over hundreds of nanoseconds, while Cer and cholesterol take around a microsecond or longer to translocate. Electron density and neutron scattering length density profiles from simulation agree with experiment, and the high disorder of linoleate in CerEOS supports experiments with infrared spectroscopy and nuclear magnetic resonance. Lateral organization demonstrates dependence on lipid composition and bilayer thickness. To further validate the LPP model, umbrella sampling was used to calculate ethanol permeability in comparison with experiment (log(P) values obtained from modeling the SC's multiple LPP layers are -7.6 and -6.6 cm/s, and that from experiment on cadaver skin is -6.65 cm/s). A "leapfrog" mechanism for ethanol permeation is proposed which is associated with its role as a topical enhancer. These models, the first experimentally verified atomistic sandwich models of the LPP, will aid in the design and optimization of transdermal drug delivery.

An In-Depth Look at DNA Crystals through the Prism of Molecular Dynamics Simulations

Summary X-ray crystallography is the primary tool for biomolecular structural determination. However, contacts formed through the crystal lattice are known to affect structures, especially for small and flexible molecules such as DNA oligomers, by introducing significant structural changes in comparison to solution. Furthermore, why molecules crystallize in certain symmetry groups, which role crystallization additives play, and whether they are just innocuous and unspecific crystallization catalysts remain unclear. By using one of the currently best-performing DNA force fields and applying significant computational effort, we described the nature of intermolecular forces that stabilize B-DNA crystals in various symmetry groups and solvent environments with an unprecedented level of detail. We showed a tight coupling between the lattice stability and the type of crystallization additives and that certain symmetry groups are stable only in the presence of a specific additive. Additives and crystal contacts induce small but non-negligible changes in the physical properties of DNA.

Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter.

ATP-binding cassette (ABC) transporters are ATP-driven molecular machines, in which ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) is chemomechanically coupled to large-scale, alternating access conformational changes in the transmembrane domains (TMDs), ultimately leading to the translocation of substrates across biological membranes. The precise nature of the structural dynamics behind the large-scale conformational transition as well as the coupling of NBD and TMD motions is still unresolved. In this work, we combine all-atom molecular dynamics (MD) simulations with electron paramagnetic resonance (EPR) spectroscopy to unravel the atomic-level mechanism of the dynamic conformational transitions underlying the functional working cycle of the heterodimeric ABC exporter TM287/288. Extensive multimicrosecond simulations in an explicit membrane/water environment show how in response to ATP binding, TM287/288 undergoes spontaneous conformational transitions from the inward-facing (IF) state via an occluded (Occ) intermediate to an outward-facing (OF) state. The latter two states have thus far not been characterized at atomic level. ATP-induced tightening of the NBD dimer involves closing and reorientation of the two NBD monomers concomitant with a closure of the intracellular TMD gate, which leads to the occluded state. Subsequently, opening at the extracellular TMD gate yields the OF conformer. The obtained mechanism imposes NBD-TMD coupling via a tight orchestration of conformational transitions, between both the two domains and also within the TMDs, ensuring that the cytoplasmic and periplasmic gate regions are never open simultaneously.

Mechanism of Large-Scale Alternating Access Conformational Transition in the ABC Exporter TM287/288

ATP-binding cassette (ABC) transporters are molecular machines that are driven by the chemo-mechanical coupling of ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) to large-scale conformational changes in the transmembrane domains (TMDs), which ultimately leads to the translocation of substrates across biological membranes. We use all-atom molecular dynamics (MD) simulations to investigate the mechanisms underlying the functional working cycle of the ABC transporter TM287/288, a prototypical heterodimeric ABC exporter. Unbiased multi-microsecond simulations in an explicit membrane/water environment reveal that in response to ATP binding, TM287/288 undergoes a spontaneous alternating-access type conformational transition from the inward-facing state seen in the x-ray crystal structure via a metastable occluded intermediate to an outward-facing state. For the latter two states, which could thus far not be structurally characterized for heterodimeric exporters, the structures obtained from our simulations are reminiscent to x-ray structures of the homologous transporters PCAT1 and Sav1866, respectively. These results allow us to describe the large-scale conformational transition underlying the working cycle of the ABC exporter in atomic detail.

Exploring DNA dynamics within oligonucleosomes with coarse-grained simulations: SIRAH force field extension for protein-DNA complexes

Describing the regulation of chromatin segments by protein recognition events constitute a major goal in biology and biotechnology. Despite astonishing experimental developments, achieving nearly atomistic spatial/temporal resolution on such macromolecular systems remains a big challenge owing to the intrinsic flexibility of large biological assemblies. Although computer simulations have become a reliable complement to experimental techniques, computational cost limits their routine applications to relatively small systems. However, the development of accurate and cost-effective coarse-grained (CG) models helps to bridge the gap between molecular dynamics simulations and biologically relevant scales.Performing an exhaustive search on a set of well-resolved crystallographic protein-DNA complexes, we introduced improvements on the CG SIRAH force field to describe protein-DNA interfaces. Modifications were validated against a set of non redundant structures and applied to the simulation of the longest DNA segment in complex with proteins that has been crystallized to date, i.e. a tetranucleosome. Multimicrosecond simulation of this small chromatin segment evidences a large mobility of the external DNA filaments, which is consistent with results from FRET experiments in solution. Moreover, we found that the sub-microsecond dynamics of DNA is strongly modulated by the quaternary structure, partially overcoming the intrinsic dynamics dictated by the primary structure.

Amyloid Beta Peptide Folding in Reverse Micelles.

Previously published experimental studies have suggested that when the 40-residue amyloid beta peptide is encapsulated in a reverse micelle, it folds into a structure that may nucleate amyloid fibril formation (Yeung, P. S.-W.; Axelsen, P. H. J. Am. Chem. Soc. 2012, 134, 6061 ). The factors that induce the formation of this structure have now been identified in a multi-microsecond simulation of the same reverse micelle system that was studied experimentally. Key features of the polypeptide-micelle interaction include the anchoring of a hydrophobic residue cluster into gaps in the reverse micelle surface, the formation of a beta turn at the anchor point that brings N- and C-terminal segments of the polypeptide into proximity, high ionic strength that promotes intramolecular hydrogen bond formation, and deformation of the reverse micelle surface to facilitate interactions with the surface along the entire length of the polypeptide. Together, these features cause the simulation-derived vibrational spectrum to red shift in a manner that reproduces the red-shift previously reported experimentally. On the basis of these findings, a new mechanism is proposed whereby membranes nucleate fibril formation and facilitate the in-register alignment of polypeptide strands that is characteristic of amyloid fibrils.

Long-timescale dynamics of the Drew-Dickerson dodecamer.

We present a systematic study of the long-timescale dynamics of the Drew–Dickerson dodecamer (DDD: d(CGCGAATTGCGC)2) a prototypical B-DNA duplex. Using our newly parameterized PARMBSC1 force field, we describe the conformational landscape of DDD in a variety of ionic environments from minimal salt to 2 M Na+Cl− or K+Cl−. The sensitivity of the simulations to the use of different solvent and ion models is analyzed in detail using multi-microsecond simulations. Finally, an extended (10 μs) simulation is used to characterize slow and infrequent conformational changes in DDD, leading to the identification of previously uncharacterized conformational states of this duplex which can explain biologically relevant conformational transitions. With a total of more than 43 μs of unrestrained molecular dynamics simulation, this study is the most extensive investigation of the dynamics of the most prototypical DNA duplex.

Modulation of a Bacterial Voltage-Gated Sodium Channel by the Anti-Epileptic Drug Lacosamide

Voltage-gated sodium channels play essential roles in electrical signaling and are primary targets in the treatment of pathologies such as epilepsy. Bacterial sodium channels are known to exhibit similar modulation by anti-epileptic agents, yet the interactions and modulation mechanisms remain unsolved at the molecular level. We have carried out multi-microsecond simulations of the bacterial NavAb channel in the presence of the drug lacosamide, a novel and effective antiepileptic drug which appears to favor slow inactivated states. We have observed drug access to the channel pore via membrane-bound fenestrations, binding to residue F203 that is a surrogate for the highly conserved FS6 in mammalian channels. We also see significant binding at the cytoplasmic gate (N211), as well as interactions with charged Arg side chains throughout entire voltage sensor domains. Calculated dissociation constants reveal that lacosamide has stronger affinity in specific conformations of the pore-forming S6 helices associated with slow inactivation and consistent with experimental evidence from mammalian channels (1). Moreover, we hypothesize that asymmetric binding within the tetrameric channel may instigate pore collapse, helping to explain this state-dependence. These studies provide new insight into sodium channel modulation, and predictions to assist in the development of improved anti-epileptic drugs.1. Errington et al (2008) Mol. Pharm., 73(1), 157-169

Comparing molecular dynamics force fields in the essential subspace.

The continued development and utility of molecular dynamics simulations requires improvements in both the physical models used (force fields) and in our ability to sample the Boltzmann distribution of these models. Recent developments in both areas have made available multi-microsecond simulations of two proteins, ubiquitin and Protein G, using a number of different force fields. Although these force fields mostly share a common mathematical form, they differ in their parameters and in the philosophy by which these were derived, and previous analyses showed varying levels of agreement with experimental NMR data. To complement the comparison to experiments, we have performed a structural analysis of and comparison between these simulations, thereby providing insight into the relationship between force-field parameterization, the resulting ensemble of conformations and the agreement with experiments. In particular, our results show that, at a coarse level, many of the motional properties are preserved across several, though not all, force fields. At a finer level of detail, however, there are distinct differences in both the structure and dynamics of the two proteins, which can, together with comparison with experimental data, help to select force fields for simulations of proteins. A noteworthy observation is that force fields that have been reparameterized and improved to provide a more accurate energetic description of the balance between helical and coil structures are difficult to distinguish from their “unbalanced” counterparts in these simulations. This observation implies that simulations of stable, folded proteins, even those reaching 10 microseconds in length, may provide relatively little information that can be used to modify torsion parameters to achieve an accurate balance between different secondary structural elements.

Ion conduction and conformational flexibility of a bacterial voltage-gated sodium channel

Voltage-gated Na(+) channels play an essential role in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. The recent solution of X-ray structures for the bacterial channel NavAb has provided an opportunity to study functional mechanisms at the atomic level. This channel's selectivity filter exhibits an EEEE ring sequence, characteristic of mammalian Ca(2+), not Na(+), channels. This raises the fundamentally important question: just what makes a Na(+) channel conduct Na(+) ions? Here we explore ion permeation on multimicrosecond timescales using the purpose-built Anton supercomputer. We isolate the likely protonation states of the EEEE ring and observe a striking flexibility of the filter that demonstrates the necessity for extended simulations to study conduction in this channel. We construct free energy maps to reveal complex multi-ion conduction via knock-on and "pass-by" mechanisms, involving concerted ion and glutamate side chain movements. Simulations in mixed ionic solutions reveal relative energetics for Na(+), K(+), and Ca(2+) within the pore that are consistent with the modest selectivity seen experimentally. We have observed conformational changes in the pore domain leading to asymmetrical collapses of the activation gate, similar to proposed inactivated structures of NavAb, with helix bending involving conserved residues that are critical for slow inactivation. These structural changes are shown to regulate access to fenestrations suggested to be pathways for lipophilic drugs and provide deeper insight into the molecular mechanisms connecting drug activity and slow inactivation.

Microsecond kinetics in model single- and double-stranded amylose polymers.

Amylose, a component of starch with increasing biotechnological significance, is a linear glucose polysaccharide that self-organizes into single- and double-helical assemblies. Starch granule packing, gelation and inclusion-complex formation result from finely balanced macromolecular kinetics that have eluded precise experimental quantification. Here, graphics processing unit (GPU) accelerated multi-microsecond aqueous simulations are employed to explore conformational kinetics in model single- and double-stranded amylose. The all-atom dynamics concur with prior X-ray and NMR data while surprising and previously overlooked microsecond helix-coil, glycosidic linkage and pyranose ring exchange are hypothesized. In a dodecasaccharide, single-helical collapse was correlated with linkages and rings transitioning from their expected syn and (4)C1 chair conformers. The associated microsecond exchange rates were dependent on proximity to the termini and chain length (comparing hexa- and trisaccharides), while kinetic features of dodecasaccharide linkage and ring flexing are proposed to be a good model for polymers. Similar length double-helices were stable on microsecond timescales but the parallel configuration was sturdier than the antiparallel equivalent. In both, tertiary organization restricted local chain dynamics, implying that simulations of single amylose strands cannot be extrapolated to dimers. Unbiased multi-microsecond simulations of amylose are proposed as a valuable route to probing macromolecular kinetics in water, assessing the impact of chemical modifications on helical stability and accelerating the development of new biotechnologies.

Breathing, bubbling, and bending: DNA flexibility from multimicrosecond simulations

Bending of the seemingly stiff DNA double helix is a fundamental physical process for any living organism. Specialized proteins recognize DNA inducing and stabilizing sharp curvatures of the double helix. However, experimental evidence suggests a high protein-independent flexibility of DNA. On the basis of coarse-grained simulations, we propose that DNA experiences thermally induced kinks associated with the spontaneous formation of internal bubbles. Comparison of the protein-induced DNA curvature calculated from the Protein Data Bank with that sampled by our simulations suggests that thermally induced distortions can account for ~80% of the DNA curvature present in experimentally solved structures.

Spontaneous Movement of Transmembrane Segments from SecY into the Membrane

At an early stage of synthesis, nearly all membrane proteins are targeted to a protein-conducting channel in the membrane, the SecY/Sec61 complex. Access of the nascent protein to the membrane occurs via a lateral gate, located at the interface of the two halves of the channel. Recently, we structurally resolved a membrane-protein insertion intermediate state of SecY bound to a translating ribosome with a transmembrane (TM) segment at the lateral gate, between channel and membrane. Beginning from that state, we have now carried out multi-microsecond simulations of different putative TM segments at SecY's lateral gate using Anton, a special-purpose molecular dynamics computer from DE Shaw Research. The simulations reveal spontaneous motion of the TM segment, either toward membrane or toward the channel interior, depending on its sequence. Diffusion of the TM segment into the membrane is generated through interactions with individual lipid molecules, which rapidly envelop a hydrophobic TM segment but reject a hydrophilic one. Subsequent free-energy calculations quantify and support the generality of the spontaneous motions observed in individual simulations. Thus, we suggest that the discrimination step between membrane-inserted and secreted states of a nascent protein occurs primarily in the SecY channel.

Microsecond Scale Replica Exchange Molecular Dynamic Simulation of Villin Headpiece: An Insight into the Folding Landscape

Reaching the experimental time scale of millisecond is a grand challenge for protein folding simulations. The development of advanced Molecular Dynamics techniques like Replica Exchange Molecular Dynamics (REMD) makes it possible to reach these experimental timescales. In this study, an attempt has been made to reach the multi microsecond simulation time scale by carrying out folding simulations on a three helix bundle protein, Villin, by combining REMD and Amber United Atom model. Twenty replicas having different temperatures ranging from 295 K to 390 K were simulated for 1.5 μs each. The lowest Root Mean Square Deviation (RMSD) structure of 2.5 Å was obtained with respect to native structure (PDB code 1VII), with all the helices formed. The folding population landscapes were built using segment-wise RMSD and Principal Components as reaction coordinates. These analyses suggest the two-stage folding for Villin. The combination of REMD and Amber United Atom model may be useful to understand the folding mechanism of various fast folding proteins