Protein under pressure: molecular dynamics simulation of the arc repressor

Experimental nuclear magnetic resonance results for the Arc Repressor have shown that this dimeric protein dissociates into a molten globule at high pressure. This structural change is accompanied by a modification of the hydrogen-bonding pattern of the intermolecular beta-sheet: it changes its character from intermolecular to intramolecular with respect to the two monomers. Molecular dynamics simulations of the Arc Repressor, as a monomer and a dimer, at elevated pressure have been performed with the aim to study this hypothesis and to identify the major structural and dynamical changes of the protein under such conditions. The monomer appears less stable than the dimer. However, the complete dissociation has not been seen because of the long timescale needed to observe this phenomenon. In fact, the protein structure altered very little when increasing the pressure. It became slightly compressed and the dynamics of the side-chains and the unfolding process slowed down. Increasing both, temperature and pressure, a tendency of conversion of intermolecular into intramolecular hydrogen bonds in the beta-sheet region has been detected, supporting the mentioned hypothesis. Also, the onset of denaturation of the separated chains was observed.     

Principles of carbopeptoid folding: a molecular dynamics simulation study.

The conformational spaces of five oligomers of tetrahydrofuran-based carbopeptoids in chloroform and dimethyl sulfoxide were investigated through nine molecular dynamics simulations. Prompted by nuclear magnetic resonance experiments that indicated various stable folds for some but not all of these carbopeptoids, their folding behaviour was investigated as a function of stereochemistry, chain length and solvent. The conformational distributions of these molecules were analysed in terms of occurrence of hydrogen bonds, backbone torsional-angle distributions, conformational clustering and solute configurational entropy. While a cis-linkage across the tetrahydrofuran ring favours right-handed helical structures, a trans-linkage results in a larger conformational variability. Intra-solute hydrogen bonding is reduced with increasing chain length and with increasing solvent polarity. Solute configurational entropies confirm the picture obtained: they are smaller for cis- than for trans-linked peptides, for chloroform than for dimethyl sulfoxide as solvent and for shorter peptide chains. The simulations provide an atomic picture of molecular conformational variability that is consistent with the available experimental data.     

The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state

We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures.     

Conformational dynamics of the mitochondrial ADP/ATP carrier: a simulation study

The mitochondrial ADP/ATP carrier is a six helix bundle membrane transport protein, which couples the exit of ATP from the mitochondrial matrix to the entry of ADP. Extended (4×20 ns) molecular dynamics simulations of the carrier, in the presence and absence of bound inhibitor (carboxyatractyloside), have been used to explore the conformational dynamics of the protein in a lipid bilayer environment, in the presence and absence of the carboxyatractyloside inhibitor. The dynamic flexibility (measured as conformational drift and fluctuations) of the protein is reduced in the presence of bound inhibitor. Proline residues in transmembrane helices H1, H3 and H5 appear to form dynamic hinges. Fluctuations in inter-helix salt bridges are also observed over the time course of the simulations. Inhibitor-protein and lipid-protein interactions have been characterised in some detail. Overall, the simulations support a transport mechanism in which flexibility about the proline hinges enables a transition between a ‘closed’ and an ‘open’ pore-like state of the carrier protein.     

Structural studies of SNARE complex and its interaction with complexin by molecular dynamics simulation

Since neurotransmitter releasing into the synaptic space delivers electrical signals from presynaptic neural cell to the postsynaptic cell, neurotransmitter secretion must be much orchestrated. Crowded intracellular vesicles involving neurotransmitters present a question of the how secretory vesicles fuse onto the plasma membrane in a fast synchronized fashion. Complexin is one of the most experimentally studied proteins that regulate assembly of fusogenic four‐helix SNARE complex to synchronized neurotransmitter secretion. We used MD simulation to investigate the interaction of complexin with the neural SNARE complex in detail. Our results show that the SNARE complex interacts with the complexin central helix by forming salt bridges and hydrogen bonds. Complexin also can interact with the Q‐SNARE complex instead of synaptobrevin to decrease the Q‐SNARE flexibility. The complexin alpha‐accessory helix and the C‐terminal region of synaptobrevin can interact with the same region of syntaxin. Although the alpha‐accessory helix aids the tight binding of the central helix to the SNARE complex, its proximity with synaptobrevin causes the destabilization of syntaxin and Sn1 helices. This study suggests that the alpha‐accessory helix of complexin can be an inhibiting factor for membrane fusion by competing with synaptobrevin for binding to the Q‐SNARE complex. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 560–570, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Evidence for lipid packaging in the crystal structure of the GM2-activator complex with platelet activating factor

GM2-activator protein (GM2-AP) is a lipid transfer protein that has the ability to stimulate the enzymatic processing of gangliosides as well as T-cell activation through lipid presentation. Our previous X-ray crystallographic studies of GM2-AP have revealed a large lipid binding pocket as the central overall feature of the structure with non-protein electron density within this pocket suggesting bound lipid. To extend these studies, we present here the 2A crystal structure of GM2-AP complexed with platelet activating factor (PAF). PAF is a potent phosphoacylglycerol whose toxic patho-physiological effects can be inhibited by GM2-AP. The structure shows an ordered arrangement of two bound lipids and a fatty acid molecule. One PAF molecule binds in an extended conformation within the hydrophobic channel that has an open and closed conformation, and was seen to contain bound phospholipid in the low pH apo structure. The second molecule is submerged inside the pocket in a U-shaped conformation with its head group near the single polar residue S141. It was refined as lyso-PAF as it lacks electron density for the sn-2 acetate group. The alkyl chains of PAF interact through van der Waals' contacts, while the head groups bind in different environments with their phosphocholine moieties in contact with aromatic rings (Y137, F80). The structure has revealed further insights into the lipid binding properties of GM2-AP, suggesting an unexpected unique mode of lipid packaging that may explain the efficiency of GM2-AP in inhibiting the detrimental biological effects of PAF.     

Analysis of crystal growth of methane hydrate using molecular dynamics simulation

Methane hydrate is a crystalline compound with methane molecules enclosed in cages formed by hydrogen-bonded water molecules. Understanding the mechanism of nucleation and crystal growth from methane vapour and liquid water is important for all hydrate applications. However, processes near the water/methane interface are still unclear. In this work, we focused on the crystal growth of methane hydrate seeds located near the water/methane interface. We performed molecular dynamics (MD) simulation and analysed the crystal growth of the hydrate seed at the interface. New cages formed in the liquid water phase were stabilised when they shared faces with the hydrate seed. We also investigated the crystal growth rate as the time development of the number of methane molecules trapped in hydrate cages, based on the trajectory of the MD simulation. The calculated growth rate in the direction that covers the interface was 1.38 times that in the direction towards the inside of the water phase.     

Catalytic mechanism of cyclophilin as observed in molecular dynamics simulations: pathway prediction and reconciliation of X-ray crystallographic and NMR solution data

Cyclophilins are proteins that catalyze X-proline cis-trans interconversion, where X represents any amino acid. Its mechanism of action has been investigated over the past years but still generates discussion, especially because until recently structures of the ligand in the cis and trans conformations for the same system were lacking. X-ray crystallographic structures for the complex cyclophilin A and HIV-1 capsid mutants with ligands in the cis and trans conformations suggest a mechanism where the N-terminal portion of the ligand rotates during the cis-trans isomerization. However, a few years before, a C-terminal rotating ligand was proposed to explain NMR solution data. In the present study we use molecular dynamics (MD) simulations to generate a trans structure starting from the cis structure. From simulations starting from the cis and trans structures obtained through the rotational pathways, the seeming contradiction between the two sets of experimental data could be resolved. The simulated N-terminal rotated trans structure shows good agreement with the equivalent crystal structure and, moreover, is consistent with the NMR data. These results illustrate the use of MD simulation at atomic resolution to model structural transitions and to interpret experimental data.     

Comparing atomistic molecular mechanics force fields for a difficult target: a case study on the Alzheimer’s amyloid β-peptide

Macromolecular function arises from structure, and many diseases are associated with misfolding of proteins. Molecular simulation methods can augment experimental techniques to understand misfolding and aggregation pathways with atomistic resolution, but the reliability of these predictions is a function of the parameters used for the simulation. There are many biomolecular force fields available, but most are validated using stably folded structures. Here, we present the results of molecular dynamics simulations on the intrinsically disordered amyloid β-peptide (Aβ), whose misfolding and aggregation give rise to the symptoms of Alzheimer’s disease. Because of the link between secondary structure changes and pathology, being able to accurately model the structure of Aβ would greatly improve our understanding of this disease, and it may facilitate application of modeling approaches to other protein misfolding disorders. To this end, we compared five popular atomistic force fields (AMBER03, CHARMM22 + CMAP, GROMOS96 53A6, GROMOS96 54A7, and OPLS-AA) to determine which could best model the structure of Aβ. By comparing secondary structure content, NMR shifts, and radius of gyration to available experimental data, we conclude that AMBER03 and CHARMM22 + CMAP over-stabilize helical structure within Aβ, with CHARMM22 + CMAP also producing elongated Aβ structures, in conflict with experimental findings. OPLS-AA, GROMOS96 53A6, and GROMOS96 54A7 produce very similar results in terms of helical and β-strand content, calculated NMR shifts, and radii of gyration that agree well with experimental data.     

Explicit-solvent molecular dynamics simulations of a DNA tetradecanucleotide duplex: lattice-sum versus reaction-field electrostatics

Five long-timescale (10 ns) explicit-solvent molecular dynamics simulations of a DNA tetradecanucleotide dimer are performed using the GROMOS 45A4 force field and the simple-point-charge water model, in order to investigate the effect of the treatment of long-range electrostatic interactions as well as of the box shape and size on the structure and dynamics of the molecule (starting from an idealised B-DNA conformation). Long-range electrostatic interactions are handled using either a lattice-sum (LS) method (particle–particle–particle–mesh; one simulation performed within a cubic box) or a cutoff-based reaction-field (RF) method (four simulations, with long-range cutoff distances of 1.4 or 2.0 nm and performed within cubic or truncated octahedral periodic boxes). The overall double-helical structure, including Watson–Crick (WC) base-pairing, is well conserved in the simulation employing the LS scheme. In contrast, the WC base-pairing is nearly completely disrupted in the four simulations employing the RF scheme. These four simulations result in highly distorted compact (cutoff distance of 1.4 nm) or extended (cutoff distance of 2 nm) structures, irrespective of the shape and size of the computational box. These differences observed between the two schemes seem correlated with large differences in the radial distribution function between charged entities (backbone phosphate groups and sodium counterions) within the system.     

FF12MC: A revised AMBER forcefield and new protein simulation protocol

Specialized to simulate proteins in molecular dynamics (MD) simulations with explicit solvation, FF12MC is a combination of a new protein simulation protocol employing uniformly reduced atomic masses by tenfold and a revised AMBER forcefield FF99 with (i) shortened C? H bonds, (ii) removal of torsions involving a nonperipheral sp³ atom, and (iii) reduced 1–4 interaction scaling factors of torsions ? and ψ. This article reports that in multiple, distinct, independent, unrestricted, unbiased, isobaric–isothermal, and classical MD simulations FF12MC can (i) simulate the experimentally observed flipping between left‐ and right‐handed configurations for C14–C38 of BPTI in solution, (ii) autonomously fold chignolin, CLN025, and Trp‐cage with folding times that agree with the experimental values, (iii) simulate subsequent unfolding and refolding of these miniproteins, and (iv) achieve a robust Z score of 1.33 for refining protein models TMR01, TMR04, and TMR07. By comparison, the latest general‐purpose AMBER forcefield FF14SB locks the C14–C38 bond to the right‐handed configuration in solution under the same protein simulation conditions. Statistical survival analysis shows that FF12MC folds chignolin and CLN025 in isobaric–isothermal MD simulations 2–4 times faster than FF14SB under the same protein simulation conditions. These results suggest that FF12MC may be used for protein simulations to study kinetics and thermodynamics of miniprotein folding as well as protein structure and dynamics. Proteins 2016; 84:1490–1516. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

Cluster analysis of hydration waters around the active sites of bacterial alanine racemase using a 2-ns MD simulation

Structural data produced by a 2-ns molecular dynamics (MD) simulation on Geobacillus alanine racemase (AlaR; PDB: 1SFT) was used to study hydration around the two AlaR active sites. AlaR is a crucial enzyme for bacterial cell wall biosynthesis. It has been shown previously that the potency of an inhibitor can be increased by incorporating a functional group or atom that displaces hydration sites close to the substrate binding pocket of its target enzyme. The complete linkage algorithm was used for cluster analysis of the active site water positions from 126 solvent configurations sampled at regular intervals from the 2-ns MD simulation. Crystal waters in the 1SFT X-ray structure occupy most of the tightly bound water sites that were discovered. We show here that tightly bound water sites can be identified by cluster analysis of MD-generated coordinates starting with data supplied by a single X-ray structure, and we predict a highly conserved hydration site close to the carboxyl oxygen of L-Ala substrate. This approach holds promise for accelerating the drug design process. We also discuss an analysis of the well-known notion of residence time and introduce a new measure called retention time.     

Molecular dynamics simulation of the dissolution process of a cellulose triacetate-II nano-sized crystal in DMSO

An understanding of the dissolution process of cellulose derivatives is important not only for basic research but also for industrial purposes. We investigated the dissolution process of cellulose triacetate II (CTA II) nano-sized crystal in DMSO solvent using molecular dynamics simulations. The nano-sized crystal consists of 18 CTA chains. During the 9 ns simulation, it was observed that one chain (C01) located at the corner of the lozenge crystal was solvated by the DMSO molecules and moved away from the remaining cluster into the DMSO solvent. The analysis showed that the breakage of the interaction between the H1, H3, and H5 hydrogens of the pyranose ring and the acetyl carbonyl oxygen in the C01 and C02 adjacent chains would be crucial for the dissolution of CTA. The DMSO molecules solvating around these atoms would prevent the re-crystallization of the CTA molecules and facilitate further dissolution.     

Membrane protein dynamics versus environment: simulations of OmpA in a micelle and in a bilayer

The bacterial outer membrane protein OmpA is one of the few membrane proteins whose structure has been solved both by X-ray crystallography and by NMR. Crystals were obtained in the presence of detergent, and the NMR structure is of the protein in a detergent micelle. We have used 10 ns duration molecular dynamics simulations to compare the behaviour of OmpA in a detergent micelle and in a phospholipid bilayer. The dynamic fluctuations of the protein structure seem to be ca 1.5 times greater in the micelle environment than in the lipid bilayer. There are subtle differences between the nature of OmpA-detergent and OmpA-lipid interactions. As a consequence of the enhanced flexibility of the OmpA protein in the micellar environment, side-chain torsion angle changes are such as to lead to formation of a continuous pore through the centre of the OmpA molecule. This may explain the experimentally observed channel formation by OmpA.     

SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation

The sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) couples ATP hydrolysis to transport of Ca²⁺. This directed energy transfer requires cross‐talk between the two Ca²⁺ sites and the phosphorylation site over 50 Å distance. We have addressed the mechano‐structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu³⁰⁹ contributes to Ca²⁺ coordination at site II, and a consensus has been that E309Q only binds Ca²⁺ at site I. The crystal structure of E309Q in the presence of Ca²⁺ and an ATP analogue, however, reveals two occupied Ca²⁺ sites of a non‐catalytic Ca₂E1 state. Ca²⁺ is bound with micromolar affinity by both Ca²⁺ sites in E309Q, but without cooperativity. The Ca²⁺‐bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A‐domain, requiring a shift of transmembrane segment M1 into an ‘up and kinked position’. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca²⁺ site II.     

A strange calmodulin of yeast

Calmodulin of Saccharomyces cerevisiae has different Ca2+ binding properties from other calmodulins. We previously reported that the maximum number of Ca2+ binding was 3 mol/mol and the fourth binding site was defective, which was different from 4 mol/mol for others. Their macroscopic dissociation constants suggested the cooperative three Ca2+ bindings rather than a pair of cooperative two Ca2+ bindings of ordinary calmodulin. Here we present evidence for yeast calmodulin showing the intramolecular close interaction between the N-terminal half domain and the C-terminal half domain, while the two domains of ordinary calmodulin are independent of each other. We will discuss the relationship of the shape and the shape change caused by the Ca2+ binding to the enzyme activation in yeast. The functional feature of calmodulin in yeast will also be considered, which might be different from the one of vertebrate calmodulin.     

Computer simulation of the association of caffeine and actinocin derivatives in aqueous solutions

The association of caffeine and actinocin derivatives (ActII), analogs of the antitumor antibiotic actinomycin D, was studied by molecular dynamics simulation in explicit solvent (water and Na⁺ and Cl^? ions). Information was obtained describing in detail the association of caffeine and ActII in water and water-salt solution and the interaction of monomers and their associates with the ionic hydrate environment. The schemes of hydration of monomers of actinocin derivatives and caffeine and their self-and heteroassociates are determined. The calculated energies of monomer interaction in associates indicate that dimerization of these compounds in aqueous solutions is advantageous in energy. Both self-and heteroassociates are stabilized by van der Waals, electrostatic, and hydrophobic interactions, as well as intermolecular hydrogen bonds. The rearrangement of the hydration shells of monomers during their association in water is energy-unfavorable and destabilizes the associates. In water-salt solutions, it is energy-favorable for the systems containing associates of the singly charged ActII⁺ and caffeine-ActII⁺. The formation of caffeine-actinocin heterodimers is preferable in energy to the formation of self-associates. In this way caffeine can decrease the concentration of the actinocin derivatives in solution and thereby decrease their biological activity.