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

Abstract

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     

Alpha7 Helix Plays an Important Role in the Conformational Stability of PTP1B

Abstract

The C-terminus of Protein Tyrosine Phosphatase 1B (PTP1B) includes an α-helix (α7), which forms an allosteric binding site 20 Å away from the active site. This helix is specific to PTP1B and its truncation decreases the catalytic activity significantly. Here, molecular dynamics (MD) simulations in the presence and absence of α7 were performed to investigate the role played by α7. The highly mobile α7 was found to maintain its contacts with loop 11 (L11)- α3 helix throughout the simulations. The interactions of Tyr152 on L11, Tyr176, Thr177 on the catalytically important WPD loop and Ser190 on α3 are important for the conformational stability and the concerted motions of the regions surrounding the WPD loop. In the absence of α7, L11 and WPD loop move away from their crystal structure conformations, resulting in the loss of the interactions in this region, and a decrease in the residue displacement correlations in the vicinity of WPD loop. Therefore, we suggest that one of the functionally important roles of α7 may be to limit the L11 and α3 motions, and, facilitate the WPD loop motions. Truncation of α7 in PTP1B is found to affect distant regions as well, such as the substrate recognition site and the phosphate binding-loop (P-loop), changing the conformations of these regions significantly. Our results show that the PTP1B specific α7 is important for the conformation and dynamics of the WPD loop, and also may play a role in ligand binding.     

Molecular Dynamics Simulations of β-turn Forming Tetra- and Hexapeptides

Abstract

It was previously shown that the structural ensemble of model peptides DDKG and GKDG (H. Ishii et al. Biopolymers 24, 2045–2056, 1985), DEKS (A. Otter et al. J. Biomol. Struct. Dyn. 7, 455–476, 1989) NPGQ (F. R. Carbone et al. Int. J. Pept. Protein. Res. 26, 498–508, 1985), SALN (H. Santa et al. J. Biomol. Struct. Dyn. 16, 1033–1041, 1999), SYPFDV and SYPYDV (J. Yao et al. J. Mol. Biol. 243, 736–753, 1994), VP^DAH and VP^DSH (B. Imperiali et al. J. Am. Chem. Soc. 114, 3182–3188, 1992) in solution contains a significant—or in some cases dominant—proportion of β-turn conformation. In this study, a protein database was searched for the above, unprotected sequences which incorporate only L-amino acid residues. Simulated annealing and 25 ns MD simulations of structures were also performed. The DSSP and STRIDE secondary structure-assigning algorithms and clustering were used to analyze trajectories and i, i+3 hydrogen bonds were also sought. The DSSP analysis showed a fluctuation between β-turn and random meander structure, although bend structures were not detected because of the insufficient length of peptide chains. This alternating trend was confirmed when the STRIDE algorithm was used to analyze trajectories, but STRIDE assigned more turn structures. The population of the strongest clusters was above 40% and the middle structures adopted β-turn structure for most sequences. These results are in good agreement with previous experimental results and support the idea of the ultra-marginal stability of turns in the absence of stabilizing long-range interactions of the neighboring segments of a polypeptide chain. However, interactions between the side-chains in tetrapeptides could also contribute to turn stability and result in unusual stability in some cases. Our observations suggest that such interactions are the consequence rather than the driving force of turn formation.     

Han ethnicity-specific type 2 diabetic treatment from traditional Chinese medicine?

Insulin-degrading enzyme (IDE) gene is one of the type 2 diabetes mellitus susceptibility genes specific to the Han Chinese population. IDE, a zinc-metalloendopeptidase, is a potential target for controlling insulin degradation. Potential lead compounds for IDE inhibition were identified from traditional Chinese medicine (TCM) through virtual screening and evaluation of their pharmacokinetic properties of absorption, distribution, metabolism, excretion, and toxicity. Molecular dynamics (MD) simulation was performed to validate the stability of complexes from docking simulation. The top three TCM compounds, dihydrocaffeic acid, isopraeroside IV, and scopolin, formed stable H–bond interactions with key residue Asn139, and were linked to active pocket residues His108, His112, and Glu189 through zinc. Torsion angle trajectories also indicated some stable interactions for each ligand with IDE. Molecular level analysis revealed that the TCM candidates might affect IDE through competitive binding to the active site and steric hindrance. Structural feature analysis reveals that high amounts of hydroxyl groups and carboxylic moieties contribute to anchor the ligand within the complex. Hence, we suggest the top three TCM compounds as potential inhibitor leads against IDE protein to control insulin degradation for type 2 diabetes mellitus.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:29     

The Stabilizing Effects of O-glycosylation on the Secondary Structural Integrity of the Designed α-loop-α motif by Molecular Dynamics Simulations

Abstract

In this study, various 400 ps molecular dynamics simulations were conducted to determine the stabilizing effect of O-glycosylation on the secondary structural integrity of the design α-loop-α motif, which has the optimal loop length of 7 Gly residues (denoted as N-A₁₆G₇A₁₆-C). In general, O-glycosylation stabilizes the structural integrity of the model peptide regardless of the length and position of glycosylation sites because it decreases the opportunity for water molecules to compete for the intramolecular hydrogen bonds. The designed peptide exhibits the highest helicity when residues 11 and 31 are replaced with Ser residues followed by O-linked with 3 galactose residues, representing the “face-to-face” glycosylation near the loop. In this case, the loop exhibits an extended conformation and several new hydrogen bonds are observed between the main chain of the loop and the galactose residues, resulting in decreasing the fluctuation and increasing the stability of the entire peptide. When the glycosylation are made close to the loop, the secondary structural integrity of the α-loop-α motif increases with the number of galactose residues. In addition, “face- to-face” glycosylation increases the structural integrity of this motif to a greater extent than “back-to-back” glycosylation. However, when the glycosylation are created away from the loop and near the N- and C-termini, no general rule is found for the stabilizing effect.     

Insight into TPMT∗23 mutation mis-folding using molecular dynamics simulation and protein structure analysis

Thiopurine S-methyltransferase (TPMT) is an important enzyme that metabolizes thiopurine drugs. This enzyme exhibits a large number of interindividual polymorphism. TPMT^?23 polymorphism has been reported in a few cases in the world in co-dominance with TPMT^?3A. The phenotype has been reported to affect enzyme activity in vivo and in vitro. Its underlying structural basis is not clarified yet. In our study, the wild type (WT) protein structure was analyzed and the amino acids bordering water channels in thiopurine sites were identified. Molecular dynamics of both the WT and TPMT^?23 mutation was carried out. In addition, the effects of this mutation, especially on the thiopurine site which is closed with a pincer like mechanism, were investigated. We focused on explaining how a locally occurred A167G substitution propagated through hydrogen bonds alteration to induce structural modification which affects both thiopurine and S-adenosylmethionine receptors. Finally, a genetic prediction of mutation functional consequences has been conducted confirming altered activity.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:20     

Molecular dynamics simulations of the thermal stability of tteRBP and ecRBP

Molecular dynamics simulations were performed for investigating the thermal stability of the extremely thermophilic Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) and the mesophilic homologous Escherichia coli ribose binding protein (ecRBP). The simulations for the two proteins were carried out under the room temperature (300?K) and the optimal activity temperature (tteRBP 375?K and ecRBP 329?K), respectively. The comparative analyses of the trajectories show that the two proteins have stable overall structures at the two temperatures; further analyses indicate that they both have strong side-chain interactions and different backbone flexibilities at the different temperatures. The tteRBP 375?K and ecRBP 329?K have stronger internal motion and higher flexibility than tteRBP 300?K and ecRBP 300?K, respectively, it is noted that the flexibility of tteRBP is much higher than that of ecRBP at the two temperatures. Therefore, tteRBP 375?K can adapt to high temperature due to its higher flexibility of backbone. Combining with the researches by Cuneo et al., it is concluded that the side-chain interactions and flexibility of backbone are both the key factors to maintain thermal stability of the two proteins.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:22     

Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant

Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71–Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71–Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71–Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95–Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α–β interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23     

Remarkable disparity in mechanical response among the extracellular domains of type I and II cadherins

Cadherins, a large family of calcium-dependent adhesion molecules, are critical for intercellular adhesion. While crystallographic structures for several cadherins show clear structural similarities, their relevant adhesive strengths vary and their mechanisms of adhesion between types I and II cadherin subfamilies are still unclear. Here, stretching of cadherins was explored experimentally by atomic force microscopy and computationally by steered molecular dynamics (SMD) simulations, where partial unfolding of the E-cadherin ectodomains was observed. The SMD simulations on strand-swapping cadherin dimers displayed similarity in binding strength, suggesting contributions of other mechanisms to explain the strength differences of cell adhesion in vivo. Systematic simulations on the unfolding of the extracellular domains of type I and II cadherins revealed diverse pathways. However, at the earliest stage, a remarkable similarity in unfolding was observed for the various type I cadherins that was distinct from that for type II cadherins. This likely correlates positively with their distinct adhesive properties, suggesting that the initial forced deformation in type I cadherins may be involved in cadherin-mediated adhesion.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:25