Comparative molecular dynamics simulation studies for determining factors contributing to the thermostability of chemotaxis protein “CheY”

Comparative molecular dynamics simulations of chemotaxis protein “CheY” from thermophilic origin Thermotoga maritima and its mesophilic counterpart Salmonella enterica have been performed for 10?ns each at 300 and 350?K, and 20?ns each at 400 and 450?K. The trajectories were analyzed in terms of different factors like root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessible surface area, H-bonds, salt bridge content, and protein–solvent interactions which indicate distinct differences between the two of them. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their optimum growth temperatures. However, at higher temperatures mesophilic protein shows increased overall flexibility than its thermophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are nonoverlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar direction of motions are observed for both proteins at 300?K. Overall, the mesophilic protein leads to increased conformational sampling of the phase space than its thermophilic counterpart. This is the first ever study of thermostability of CheY protein homologs by using protein dynamism as a main impact. Our study might be used as a model for studying the molecular basis of thermostability of two homologous proteins from two organisms living at different temperatures with less visible differences.     

The function and structural influence of selective relaxed constraint at functional intracellular loop3 of 5-HT1A serotonin-1 receptor family

Serotonin (5-HT) and its receptors have been involved in critical signal transduction mechanism and deregulation implicated in mood-related disorders. 5-HT activities are mediated through a family of transmembrane spanning serotonin receptors. Both within the family and species, 5-HT receptor protein sequence diversity and 7-transmembrane structural homogeneity have long been intriguing. In this study, we have analyzed the codon site constraint in 5-HT1 subclass receptors from 13 orthologous mammalian mRNA coding sequence. Further, the study was extended to computationally investigate the impact of non-synonymous sites with respect to function and structural significance through sequence homology algorithm and molecular dynamics simulation (MDS). Codon sites with significant posterior probability were observed in 5-HT_1A, 5‐HT_1B and 5-HT_1D receptor indicating variations in site constraint within the 5‐HT1 sub-class genes. In 5-HT_1A receptor, seven sites were detected at the functional intracellular loop₃ (ICL₃) with higher substitution rate through Codeml program. Sequence homology algorithm identifies that these sites were functionally tolerant within the mammals representing a selectively relaxed constraint at this domain. On the other hand, the root mean square deviation (rmsd) values from MDS suggest differences in structural conformation of ICL₃ models among the species. Specifically, the human ICL₃ model fluctuation was comparatively more stable than other species. Hence, we argue that these sites may have varying influence in G-proteins coupling and activation of effectors systems through downstream interacting accessory proteins of cell among the species. However, further experimental studies are required to elucidate the precise role and the seeming difference of these sites in 5-HT receptors between species.     

Influence of GTP/GDP and magnesium ion on the solvated structure of the protein FtsZ: a molecular dynamics study

We present here a structural analysis of ten extensive all-atom molecular dynamics simulations of the monomeric protein FtsZ in various binding states. Since the polymerization and GTPase activities of FtsZ depend on the nature of a bound nucleotide as well as on the presence of a magnesium ion, we studied the structural differences between the average conformations of the following five systems: FtsZ-Apo, FtsZ-GTP, FtsZ-GDP, FtsZ-GTP-Mg, and FtsZ-GDP-Mg. The in silico solvated average structure of FtsZ-Apo significantly differs from the crystallographic structure 1W59 of FtsZ which was crystallized in a dimeric form without nucleotide and magnesium. The simulated Apo form of the protein also clearly differs from the FtsZ structures when it is bound to its ligand, the most important discrepancies being located in the loops surrounding the nucleotide binding pocket. The three average structures of FtsZ-GTP, FtsZ-GDP, and FtsZ-GTP-Mg are overall similar, except for the loop T7 located at the opposite side of the binding pocket and whose conformation in FtsZ-GDP notably differs from the one in FtsZ-GTP and FtsZ-GTP-Mg. The presence of a magnesium ion in the binding pocket has no impact on the FtsZ conformation when it is bound to GTP. In contrast, when the protein is bound to GDP, the divalent cation causes a translation of the nucleotide outwards the pocket, inducing a significant conformational change of the loop H6-H7 and the top of helix H7.     

Homology modeling and molecular dynamics simulations of Dengue virus NS2B/NS3 protease: insight into molecular interaction

The pathogenic West Nile virus (WNV) and Dengue virus (DV) are growing global threats for which there are no specific treatments. Both viruses possess a two component NS2B/NS3 protease which cleaves viral precursor proteins. Whereas for the WNV protease two crystal structures in complex with an inhibitor have been solved recently, no such information is available for the DV protease. Here, we report the generation of a homology model of DV NS2B/NS3 protease. Since it is known from the related WNV protease that it adopts a distinct conformation in free and in inhibitor‐complexed form, a special emphasis was given to the analysis of the protease flexibility. Therefore, several models of DV NS2B/NS3 protease complexed with the peptidic inhibitor (Bz‐Nle(P4)‐Lys(P3)‐Arg(P2)‐Arg(P1)‐H) were generated. The first DV protease model (DV‐1) was constructed using the available crystal structure of the apo DV NS2B/NS3 protease. The second model (DV‐2) was built taking the WNV NS3/NS2B protease in the inhibitor‐complexed form as the template structure. Molecular dynamics simulations which were carried out for the WNV crystal structures as well as for the DV models provided an understanding of the role of NS2B for maintaining the protease in the active conformation. It was also demonstrated that NS2B is not only important for maintaining NS3 in the active form, but is also essential for establishing the interaction between residues from the S2 pocket and the peptidic inhibitor. The DV NS2B/NS3 model in the productive conformation can now be used for structure‐based design purposes. Copyright © 2009 John Wiley & Sons, Ltd.     

The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers

Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental samples when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1–2.0?mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50?mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10?mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20?mol% cholesterol not only modulated the fluidity of the bilayers; it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.     

Structural insight into the antagonistic action of diarylheptanoid on human estrogen receptor alpha

Estrogen receptor α (ER α) is an important therapeutic target in the regulation of ligand dependent signaling in breast cancer. The current study investigates the anti-estrogenic potential of the Diarylheptanoid, 5-hydroxy-7-(4-hydroxy-3 methoxyphenyl)-1-phenyl-3-heptanone (DAH) in silico. Rigid Docking analysis of DAH at the ligand binding domain (LBD) of ER α showed hydrogen bond interactions with Arg394 and Glu353 at the active site, similar to the positive controls 4-Hydroxy Tamoxifen (4-OHT) and Fulvestrant (FUL). The protein and the protein–DAH complexes were further analyzed using molecular dynamics simulations for a time scale of 50 ns using GROMACS. Root mean square fluctuation (RMSF) analysis showed large fluctuations at the N-terminal region of Helices (H) 3, 9 and at the C-terminal region of H11, which could be involved in the antagonistic conformational change. Interestingly, H12 appeared to move away from the ligand binding pocket and occupy the co-activator binding groove at the LBD of ER α. Secondary structure analysis of the protein upon binding of DAH and CUR showed structural change from α-helix to Turn conformation at H4. We hypothesize that this structural change at H4, similar to the positive control, could hinder the activity of AF-2 by blocking the binding of co-activator. These conformational changes in ER α indicate an anti-estrogenic and therapeutic potential of the DAH.     

The structural properties of magainin in water, TFE/water, and aqueous urea solutions: molecular dynamics simulations

Here, the MD simulations and comparative structural analysis of Magainin in water, TFE/water, and 2M, 4M, and BM urea solutions is reported. For MAG-TFE/water and MAG-2M urea the largely alpha helical conformation of the peptide is maintained throughout the 9-ns simulation. While in water, 4M urea, and 8M urea, the helix length decreases and at the same time helix radius increases. This suggests a more destabilized magainin secondary structure. Our simulation data reveals that the stabilizing effect of TFE is induced by preferential accumulation of TFE molecules around the alpha helical peptide. These results indicate that an aqueous urea solution solvates the surface of polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the peptide in comparison with water, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the peptide. At 8M urea, there are more direct interactions between the urea and solute, and the helix is destabilized. At 2M urea, the interaction of urea molecules and nonpolar residues are weak, therefore, the presence of urea molecules decreases the interactions of water molecules with hydrophilic groups. Urea could not deteriorate the peptide secondary structure with time from an initial helix structure.     

Computational exploration of polymorphisms in 5-hydoxytryptamine 5-HT₁A and 5-HT₂A receptors associated with psychiatric disease

The huge polymorphic data have been prioritized towards a specific disease based on sequence and structure homology tools to a large extent. In this study, we have explored the potential non-synonymous Single Nucleotide Polymorphism (nsSNP) in serotonin (5-HT) receptors involved in psychotic syndromes and their response pathway. The most damaging point mutations were screened from 12 classes of serotonin receptors comprising 7743 variants. In 5HT(1A) receptor, two alleles were found to be highly deleterious located at ligand binding extracellular-2 and one at intracellular loop-3 domains. Similarly, we found two alleles predicted to be highly damaging in 5HT(2A) residing at N and C-Terminal domains. The above alleles were further confirmed based on their flexibility and stability difference using the molecular dynamic simulation analysis. Integrating these results appeared promising for being able to filter out potential non-synonymous Single Nucleotide Polymorphisms for neuropsychiatric disorders.     

Effect of lipid composition on buforin II structure and membrane entry

Buforin II is a 21-amino acid polycationic antimicrobial peptide derived from a peptide originally isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. It is hypothesized to target a wide range of bacteria by translocating into cells without membrane permeabilization and binding to nucleic acids. Previous research found that the structure and membrane interactions of buforin II are related to lipid composition. In this study, we used molecular dynamics (MD) simulations along with lipid vesicle experiments to gain insight into how buforin II interacts differently with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) lipids. Fluorescent spectroscopic measurements agreed with the previous assertion that buforin II does not interact with pure PC vesicles. Nonetheless, the reduced entry of the peptide into anionic PG membranes versus neutral PC membranes during simulations correlates with the experimentally observed reduction in BF2 translocation through pure PG membranes. Simulations showing membrane entry into PC also provide insight into how buforin II may initially penetrate cell membranes. Our MD simulations also allowed us to consider how neutral PE lipids affect the peptide differently than PC. In particular, the peptide had a more helical secondary structure in simulations with PE lipids. A change in structure was also apparent in circular dichroism measurements. PE also reduced membrane entry in simulations, which correlates with decreased translocation in the presence of PE observed in previous studies. Together, these results provide molecular-level insight into how lipid composition can affect buforin II structure and function and will be useful in efforts to design peptides with desired antimicrobial and cell-penetrating properties.     

Reproducible polypeptide folding and structure prediction using molecular dynamics simulations

The folding of a polypeptide from an extended state to a well-defined conformation is studied using microsecond classical molecular dynamics (MD) simulations and replica exchange molecular dynamics (REMD) simulations in explicit solvent and in vacuo. It is shown that the solvated peptide folds many times in the REMD simulations but only a few times in the conventional simulations. From the folding events in the classical simulations we estimate an approximate folding time of 1-2 micros. The REMD simulations allow enough sampling to deduce a detailed Gibbs free energy landscape in three dimensions. The global minimum of the energy landscape corresponds to the native state of the peptide as determined previously by nuclear magnetic resonance (NMR) experiments. Starting from an extended state it takes about 50 ns before the native structure appears in the REMD simulations, about an order of magnitude faster than conventional MD. The calculated melting curve is in good qualitative agreement with experiment. In vacuo, the peptide collapses rapidly to a conformation that is substantially different from the native state in solvent.