Insights into internal dynamics of 6-phosphogluconolactonase from Trypanosoma brucei studied by nuclear magnetic resonance and molecular dynamics

Nuclear magnetic resonance is used to investigate the backbone dynamics in 6-phosphogluconolactonase from Trypanosoma brucei (Tb6PGL) with (holo-) and without (apo-) 6-phosphogluconic acid as ligand. Relaxation data were analyzed using the model-free approach and reduced spectral density mapping. Comparison of predictions, based on 77 ns molecular dynamics simulations, with the observed relaxation rates gives insight into dynamical properties of the protein and their alteration on ligand binding. Data indicate dynamics changes in the vicinity of the binding site. More interesting is the presence of perturbations located in remote regions of this well-structured globular protein in which no large-amplitude motions are involved. This suggests that delocalized changes in dynamics that occur upon binding could be a general feature of protein-target interactions.     

Structure, dynamics, and elasticity of free 16s rRNA helix 44 studied by molecular dynamics simulations

Molecular dynamics (MD) simulations were employed to investigate the structure, dynamics, and local base-pair step deformability of the free 16S ribosomal helix 44 from Thermus thermophilus and of a canonical A-RNA double helix. While helix 44 is bent in the crystal structure of the small ribosomal subunit, the simulated helix 44 is intrinsically straight. It shows, however, substantial instantaneous bends that are isotropic. The spontaneous motions seen in simulations achieve large degrees of bending seen in the X-ray structure and would be entirely sufficient to allow the dynamics of the upper part of helix 44 evidenced by cryo-electron microscopic studies. Analysis of local base-pair step deformability reveals a patch of flexible steps in the upper part of helix 44 and in the area proximal to the bulge bases, suggesting that the upper part of helix 44 has enhanced flexibility. The simulations identify two conformational substates of the second bulge area (bottom part of the helix) with distinct base pairing. In agreement with nuclear magnetic resonance (NMR) and X-ray studies, a flipped out conformational substate of conserved 1492A is seen in the first bulge area. Molecular dynamics (MD) simulations reveal a number of reversible alpha-gamma backbone flips that correspond to transitions between two known A-RNA backbone families. The flipped substates do not cumulate along the trajectory and lead to a modest transient reduction of helical twist with no significant influence on the overall geometry of the duplexes. Despite their considerable flexibility, the simulated structures are very stable with no indication of substantial force field inaccuracies.     

Nucleotide-mediated conformational changes of monomeric actin and Arp3 studied by molecular dynamics simulations

Members of the actin family of proteins exhibit different biochemical properties when ATP, ADP-P_i, ADP, or no nucleotide is bound. We used molecular dynamics simulations to study the effect of nucleotides on the behavior of actin and actin-related protein 3 (Arp3). In all of the actin simulations, the nucleotide cleft stayed closed, as in most crystal structures. ADP was much more mobile within the cleft than ATP, despite the fact that both nucleotides adopt identical conformations in actin crystal structures. The nucleotide cleft of Arp3 opened in most simulations with ATP, ADP, and no bound nucleotide. Deletion of a C-terminal region of Arp3 that extends beyond the conserved actin sequence reduced the tendency of the Arp3 cleft to open. When the Arp3 cleft opened, we observed multiple instances of partial release of the nucleotide. Cleft opening in Arp3 also allowed us to observe correlated movements of the phosphate clamp, cleft mouth, and barbed-end groove, providing a way for changes in the nucleotide state to be relayed to other parts of Arp3. The DNase binding loop of actin was highly flexible regardless of the nucleotide state. The conformation of Ser14/Thr14 in the P1 loop was sensitive to the presence of the γ-phosphate, but other changes observed in crystal structures were not correlated with the nucleotide state on nanosecond timescales. The divalent cation occupied three positions in the nucleotide cleft, one of which was not previously observed in actin or Arp2/3 complex structures. In sum, these simulations show that subtle differences in structures of actin family proteins have profound effects on their nucleotide-driven behavior.     

Retinal release from opsin in molecular dynamics simulations

The crystal structures of opsin in the ligand-free and the G-protein-interacting states showed two inter-helical openings between transmembrane (TM) helices TM1 and TM7 and between TM5 and TM6 near the extracellular side that were thought to serve as the retinal uptake and release gates. However, it is unclear which opening is for 11-cis-retinal uptake or all-trans-retinal release although speculations have been proposed based on the structural features of opsin and retinal. In this work, we simulated the exit process of all-trans-retinal from the ligand-free opsin structure by the classical molecular dynamics (MD) and random acceleration molecular dynamics (RAMD). In the 64 ns classical MD simulation, retinal remained in the receptor but moved significantly toward the TM5-TM6 opening and almost inserted into the opening after 50 ns. Complete exit was observed in 114 out of 160 RAMD trajectories with the TM5-TM6 opening being the predominant egress gate while egress from the TM1-TM7 opening was observed in only a few trajectories when relatively large acceleration was applied and large structural alteration of the protein resulted. These results suggest that photolyzed all-trans-retinal is likely released through the TM5-TM6 opening. Based on the unidirectional mechanism of retinal exchange suggested by experiment, we speculate that the TM1-TM7 opening serves as the 11-cis-retinal uptake gate. The spatial occupancy maps of retinal computed from the 160 RAMD trajectories further indicated that retinal experienced significant interactions with the receptor during the exit process. The implications of these findings for disease mechanisms of rhodopsin mutants are discussed.     

Ligand unbinding pathways from the vitamin D receptor studied by molecular dynamics simulations

Molecular dynamics simulation techniques have been used to study the unbinding pathways of 1α,25-dihydroxyvitamin D₃ from the ligand-binding pocket of the vitamin D receptor (VDR). The pathways observed in a large number of relatively short (<200 ps) random acceleration molecular dynamics (RAMD) trajectories were found to be in fair agreement, both in terms of pathway locations and deduced relative preferences, compared to targeted molecular dynamics (TMD) and streered molecular dynamics simulations (SMD). However, the high-velocity ligand expulsions of RAMD tend to favor straight expulsion trajectories and the observed relative frequencies of different pathways were biased towards the probability of entering a particular exit channel. Simulations indicated that for VDR the unbinding pathway between the H1–H2 loop and the β-sheet between H5 and H6 is more favorable than the pathway located between the H1–H2 loop and H3. The latter pathway has been suggested to be the most likely unbinding path for thyroid hormone receptors (TRs) and a likely path for retinoic acid receptor. Ligand entry/exit through these two pathways would not require displacement of H12 from its agonistic position. Differences in the packing of the H1, H2, H3 and β-sheet region explain the changed relative preference of the two unbinding pathways in VDR and TRs. Based on the crystal structures of the ligand binding domains of class 2 nuclear receptors, whose members are VDR and TRs, this receptor class can be divided in two groups according to the packing of the H1, H2, H3 and β-sheet region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Insight into the key active sites of afChiA1 based on molecular dynamics simulations and free energy calculations

In this study, the binding of the enzyme chitinase A1 (afChiA1) from the plant-type Aspergillus fumigatus with four potent inhibitors, allosamidin (ASM), acetazolamide (AZM), 8-chloro-theophylline (CTP) and kinetin (KIT) is investigated by molecular docking, molecular dynamics simulation and binding free energy calculation. The results reveal that the electrostatic interactions play an important role in the stabilisation of the binding of afChiA1 with inhibitors. Based on the binding energy of afChiA1-ligands, the key residues (Gln37 and Trp312) in the active binding pocket of the complex systems are confirmed by molecular mechanics/Poisson–Boltzmann surface area method, and the active inhibitors, ASM and AZM, both could form strong interaction with Gln37 and Trp312, and the non-active ligands, CTP and KIT, could not interact with these two residues, which is consistent with the result of experimental report. Then, it is identified that Gln37 and Trp312 should be one of the important active site residues of afChiA1.     

Dynamics of Ca2+-saturated calmodulin D129N mutant studied by multiple molecular dynamics simulations

Fifteen independent 1-nsec MD simulations of fully solvated Ca(2+) saturated calmodulin (CaM) mutant D129N were performed from different initial conditions to provide a sufficient statistical basis to gauge the significance of observed dynamical properties. In all MD simulations the four Ca(2+) ions remained in their binding sites, and retained a single water ligand as observed in the crystal structure. The coordination of Ca(2+) ions in EF-hands I, II, and III was sevenfold. In EF-hand IV, which was perturbed by the mutation of a highly conserved Asp129, an anomalous eightfold Ca(2+) coordination was observed. The Ca(2+) binding loop in EF-hand II was observed to dynamically sample conformations related to the Ca(2+)-free form. Repeated MD simulations implicate two well-defined conformations of Ca(2+) binding loop II, whereas similar effect was not observed for loops I, III, and IV. In 8 out of 15 MD simulations Ca(2+) binding loop II adopted an alternative conformation in which the Thr62 >C=O group was displaced from the Ca(2+) coordination by a water molecule, resulting in the Ca(2+) ion ligated by two water molecules. The alternative conformation of the Ca(2+) binding loop II appears related to the "closed" state involved in conformational exchange previously detected by NMR in the N-terminal domain fragment of CaM and the C-terminal domain fragment of the mutant E140Q. MD simulations suggest that conformations involved in microsecond exchange exist partially preformed on the nanosecond time scale.     

A statistical approach to the interpretation of molecular dynamics simulations of calmodulin equilibrium dynamics

A sample of 35 independent molecular dynamics (MD) simulations of calmodulin (CaM) equilibrium dynamics was prepared from different but equally plausible initial conditions (20 simulations of the wild-type protein and 15 simulations of the D129N mutant). CaM's radius of gyration and backbone mean-square fluctuations were analyzed for the effect of the D129N mutation, and simulations were compared with experiments. Statistical tests were employed for quantitative comparisons at the desired error level. The computational model predicted statistically significant compaction of CaM relative to the crystal structure, consistent with the results of small-angle X-ray scattering (SAXS) experiments. This effect was not observed in several previously reported studies of (Ca2+)(4)-CaM, which relied on a single MD run. In contrast to radius of gyration, backbone mean-square fluctuations showed a distinctly non-normal and positively skewed distribution for nearly all residues. Furthermore, the D129N mutation affected the backbone dynamics in a complex manner and reduced the mobility of Glu123, Met124, Ile125, Arg126, and Glu127 located in the adjacent alpha-helix G. The implications of these observations for the comparisons of MD simulations with experiments are discussed. The proposed approach may be useful in studies of protein equilibrium dynamics where MD simulations fall short of properly sampling the conformational space, and when the comparison with experiments is affected by the reproducibility of the computational model.     

Molecular dynamics simulation of interleukin-2 and its complex and determination of the binding free energy

Interleukin-2 (IL-2) protein belongs to the signal modulator cytokine's family and therefore it is prevalent for immunological responses. It has been identified as a centrally important potential drug target for the inhibition of protein-protein interactions; so as to suppress the immunological responses associated with autoimmune, inflammatory and immunological diseases, and cancer. In the present work, we have performed two independent 100?ns of molecular dynamics (MD) simulations on the apo IL-2 protein and its ligand-bound complex (with a potent inhibitor FRG), to study the effect of inhibitor binding on the dynamics and stability of the protein. The calculation of binding free energy via post-processing end state method of Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics Generalised Born Surface Area (MM-GBSA) has inferred a good correlation in accordance with the already reported experimental data, demonstrating that the free energy of binding calculated by the two methods has no significant difference. The investigation of individual components of free energy revealed that the association of IL-2 protein with FRG ligand is primarily driven by the van der Waals energy contribution that represents the non-polar/hydrophobic energy contribution as dominant in this case of ligand binding.     

Unraveling the role of flexible coil near calcium binding site of levansucrase on thermostability and product profile via proline substitution and molecular dynamics simulations

Due to its bioactivity and versatile applications, levan has appeared as a promising biomaterial. Levansucrase is responsible for the conversion of sucrose into levan. With the goal of enhancing levan production, the strategy for enhancing the stability of levansucrase is being intensively studied. To make proteins more stable under high temperatures, proline, the most rigid residue, can be introduced into previously flexible regions. Herein, G249, D250, N251, and H252 on the flexible coil close to the calcium binding site of Bacillus licheniformis levansucrase were replaced with proline. Mutations at G249P greatly enhance both the enzyme's thermodynamic and kinetic stability, while those at H252P improve solely the enzyme's kinetic stability. GPC analysis revealed that G249P synthesize more levan, but H252P generate primarily oligosaccharides. Molecular dynamics simulations (MD) and MM/GBSA analysis revealed that G249P mutation increased not only the stability of levansucrase, but also affinity toward fructan.     

Loop motion and base release in purine-specific nucleoside hydrolase: A molecular dynamics study

Although various Trypanosoma vivax purine-specific inosine–adenosine–guanosine nucleoside hydrolase (IAG-NH) crystal structures have been determined and the chemical reaction mechanism of substrate hydrolysis has been studied recently, the mechanistic details for the release of base and ribose are still unclear. Herein molecular dynamics (MD) simulations combined with umbrella sampling technique were utilized to explore the regulation mechanisms of key residues and loops 1 and 2 for the base release. Our results have indicated that the base release process is not the rate-limiting step in the entire hydrolysis process, and the very low barrier of ~ 5.6 kcal/mol can be washed out easily by the notable exothermicity from the substrate hydrolysis step. Moreover, the MD simulations have revealed that Glu82/Trp83 in loop 1 and His247/Arg252 in loop 2 are important to modulate the base release. The partial helix-to-coil change of loop 2 along with the base release process has been observed, showing good agreement with the IAG-NH crystal structures. The local binding site around the ribose after the base release is also discussed.     

Effects of ligand binding on the dynamics of rice nonspecific lipid transfer protein 1: a model from molecular simulations

Plant nonspecific lipid transfer proteins (nsLTPs) are small, basic proteins constituted mainly of alpha-helices and stabilized by four conserved disulfide bridges. They are characterized by the presence of a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers in vitro. In this study, molecular dynamics (MD) simulations were performed at room temperature to investigate the effects of lipid binding on the dynamic properties of rice nsLTP1. Rice nsLTP1, either in the free form or complexed with one or two lipids was subjected to MD simulations. The C-terminal loop was very flexible both before and after lipid binding, as revealed by calculating the root-mean-square fluctuation. After lipid binding, the flexibility of some residues that were not in direct contact with lipid molecules increased significantly, indicating an increase of entropy in the region distal from the binding site. Essential dynamics analysis revealed clear differences in motion between unliganded and liganded rice nsLTP1s. In the free form of rice nsLTP1, loop1 exhibited the largest directional motion. This specific essential motion mode diminished after binding one or two lipid molecules. To verify the origin of the essential motion observed in the free form of rice nsLTP1, we performed multiple sequence alignments to probe the intrinsic motion encoded in the primary sequence. We found that the amino acid sequence of loop1 is highly conserved among plant nsLTP1s, thus revealing its functional importance during evolution. Furthermore, the sequence of loop1 is composed mainly of amino acids with short side chains. In this study, we show that MD simulations, together with essential dynamics analysis, can be used to determine structural and dynamic differences of rice nsLTP1 upon lipid binding.     

Searching the conformational complexity and binding properties of HDAC6 through docking and molecular dynamic simulations

Histone deacetylases (HDACs) are a family of proteins involved in the deacetylation of histones and other non-histones substrates. HDAC6 belongs to class II and shares similar biological functions with others of its class. Nevertheless, its three-dimensional structure that involves the catalytic site remains unknown for exploring the ligand recognition properties. Therefore, in this contribution, homology modeling, 100-ns-long Molecular Dynamics (MD) simulation and docking calculations were combined to explore the conformational complexity and binding properties of the catalytic domain 2 from HDAC6 (DD2-HDAC6), for which activity and affinity toward five different ligands have been reported. Clustering analysis allowed identifying the most populated conformers present during the MD simulation, which were used as starting models to perform docking calculations with five DD2-HDAC6 inhibitors: Cay10603 (CAY), Rocilinostat (RCT), Tubastatin A (TBA), Tubacin (TBC), and Nexturastat (NXT), and then were also submitted to 100-ns-long MD simulations. Docking calculations revealed that the five inhibitors bind at the DD2-HDAC6 binding site with the lowest binding free energy, the same binding mode is maintained along the 100-ns-long MD simulations. Overall, our results provide structural information about the molecular flexibility of apo and holo DD2-HDAC6 states as well as insight of the map of interactions between DD2-HDAC6 and five well-known DD2-HDAC6 inhibitors allowing structural details to guide the drug design. Finally, we highlight the importance of combining different theoretical approaches to provide suitable structural models for structure-based drug design.     

Conformational changes of the p53-binding cleft of MDM2 revealed by molecular dynamics simulations

Two 35-ns molecular dynamics simulations of both ligated [mouse double minute protein 2 (MDM2(p53))] and unligated (MDM2(apo)) structures of human MDM2 bound to the N-terminal domain of the tumor suppressor p53 have been performed. Analysis of the dynamics revealed that the most flexible region of MDM2 was the p53-binding cleft. When MDM2 was bound to p53, a wider and more stable topology of the cleft was obtained, while unligated MDM2 showed a narrower and highly flexible cleft. It was also found that the dynamics involved in the opening/closing motions were due to the movement of different domains of the protein, which is in agreement with recent experimental data. Considering our results, a mechanism in which p53 might be recognized and attached to MDM2 is proposed, and some implications on future directions for in silico anticancer drug design efforts are discussed. In summary, the observations made here would be very useful not only for better understanding of the biological implications of the MDM2 dynamics, but also for future efforts in anticancer drug design and discovery.     

Influence of point mutations on the flexibility of cytochrome b5: molecular dynamics simulations of holoproteins

Two membrane-bound isoforms of cytochrome b5 have been identified in mammals, one associated with the outer mitochondrial membrane (OM b5) and the other with the endoplasmic reticulum (microsomal, or Mc b5). The soluble heme binding domains of OM and Mc b5 have highly similar three-dimensional structures but differ significantly in physical properties, with OM b5 exhibiting higher stability due to stronger heme association. In this study, we present results of 8.5-ns length molecular dynamics simulations for rat Mc b5, bovine Mc b5, and rat OM b5, as well as for two rat OM b5 mutants that were anticipated to exhibit properties intermediate between those of rat OM b5 and the two Mc proteins: the A18S/I32L/L47R triple mutant (OM3M) and the A18S/I25L/I32L/L47R/L71S quintuple mutant (OM5M). Analysis of the structure, fluctuations, and interactions showed that the five b5 variants used in this study differed in organization of their molecular surfaces and heme binding cores in a way that could be used to explain certain experimentally observed physical differences. Overall, our simulations provided qualitative microscopic explanations of many of the differences in physical properties between OM and Mc b5 and two mutants in terms of localized changes in structure and flexibility. They also reveal that opening of a surface cleft between hydrophobic cores 1 and 2 in bovine Mc b5, observed in two previously reported simulations (E. M. Storch and V. Daggett, Biochemistry, 1995, Vol. 34, pp. 9682-9693; A. Altuve, Biochemistry, 2001, Vol. 40, pp. 9469-9483), probably resulted from removal of crystal contacts and likely does not occur on the nanosecond time scale. Finally, the MD simulations of OM5M b5 verify that stability and dynamic properties of cytochrome b5 are remarkably resistant to mutations that dramatically alter the stability and structure of the apoprotein.