Protein conformational landscapes: Energy minimization and clustering of a long molecular dynamics trajectory

Using energy minimization and cluster analysis, we have analyzed a 1020 ps molecular dynamics trajectory of solvated bovine pancreatic trypsin inhibitor. Elucidation of conformational sub states in this way both illustrates the degree of conformational convergence in the simulation and reduces the structural data to a tractable subset. The relative movement of structures upon energy minimization was used to estimate the sizes of features on the protein potential energy surface. The structures were analyzed using their pairwise root-mean-square C_α deviations, which gave a global measure of conformational changes that would not be apparent by monitoring single degrees of freedom. At time scales of 0.1 ps, energy minimization detected sharp transitions between energy minima separated by 0.1 Å rms deviation. Larger conformational clusters containing these smaller minima and separated by 0.25 Å were seen at 1 ps time scales. Both of these small features of the conformational landscape were characterized by movements in loop regions associated with small, correlated backbone dihedral angle shifts. On a nanosecond time scale, the main features of the protein energy landscape were clusters separated by over 0.7 Å rms deviation, with only seven of these sub states visited over the 1 ns trajectory. These substates, discernible both before and after energy minimization, differ mainly in a monotonic pivot of the loop residues 11–18 over the course of the simulation. This loop contains lysine 17, which specifically binds to trypsin in the active site. The trajectory did not return to previously visited clusters, indicating that this trajectory has not been shown to have completely sampled the conformational substates available to it. Because the apparent convergence to a single region of conformation space depends on both the time scale of observation and the size of the conformational features examined, convergence must be operationally defined within the context of the simulation. © 1995 Wiley-Liss, Inc.     

Computer simulation as a tool for tracing the conformational differences between proteins in solution and in the crystalline state

Knowledge about the architecture of macromolecules has been derived primarily from crystallography. Therefore, it has been a matter of concern whether the conformation of a macromolecule in solution, namely in vivo, might be different from that in the crystalline state. To determine the difference between the conformations, a protein (trypsin inhibitor) dissolved in water has been simulated using the method of molecular dynamics and the results are compared with those obtained from a simulation of the full crystalline unit cell. We report here that no significant difference was found for backbone atoms, except for two more or less flexible loops extending from the core of the protein and the very flexible carboxyterminal residues. The side-chains in which the conformation in solution differs considerably from that in the crystal all belong to polar residues.     

Early events in the disulfide-coupled folding of BPTI.

Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the [14-38] intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfide-formation steps were carried out. The results of these studies indicate that the formation of [14-38] from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 degrees C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14-38] or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of [14-38] did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.     

Variability of conformations at crystal contacts in BPTI represent true low-energy structures: correspondence among lattice packing and molecular dynamics structures.

The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.     

Conformational insights on the molecular recognition processes of carbohydrate molecules by proteins and enzymes: A 3D view by using NMR

This review focuses, in a non-exhaustive manner, on the essential structural and conformational features of protein–carbohydrate interactions and on some applications of NMR spectroscopy to deal with this topic from different levels of complexity.     

Molecular dynamics studies of a DNA-binding protein: 2. An evaluation of implicit and explicit solvent models for the molecular dynamics simulation of the Escherichia coli trp repressor.

Although aqueous simulations with periodic boundary conditions more accurately describe protein dynamics than in vacuo simulations, these are computationally intensive for most proteins. Trp repressor dynamic simulations with a small water shell surrounding the starting model yield protein trajectories that are markedly improved over gas phase, yet computationally efficient. Explicit water in molecular dynamics simulations maintains surface exposure of protein hydrophilic atoms and burial of hydrophobic atoms by opposing the otherwise asymmetric protein-protein forces. This properly orients protein surface side chains, reduces protein fluctuations, and lowers the overall root mean square deviation from the crystal structure. For simulations with crystallographic waters only, a linear or sigmoidal distance-dependent dielectric yields a much better trajectory than does a constant dielectric model. As more water is added to the starting model, the differences between using distance-dependent and constant dielectric models becomes smaller, although the linear distance-dependent dielectric yields an average structure closer to the crystal structure than does a constant dielectric model. Multiplicative constants greater than one, for the linear distance-dependent dielectric simulations, produced trajectories that are progressively worse in describing trp repressor dynamics. Simulations of bovine pancreatic trypsin were used to ensure that the trp repressor results were not protein dependent and to explore the effect of the nonbonded cutoff on the distance-dependent and constant dielectric simulation models. The nonbonded cutoff markedly affected the constant but not distance-dependent dielectric bovine pancreatic trypsin inhibitor simulations. As with trp repressor, the distance-dependent dielectric model with a shell of water surrounding the protein produced a trajectory in better agreement with the crystal structure than a constant dielectric model, and the physical properties of the trajectory average structure, both with and without a nonbonded cutoff, were comparable.     

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic-->leucine substitutions on the distribution of folding intermediates.

The roles of aromatic residues in determining the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) were analyzed mutationally by examining the distribution of disulfide-bonded intermediates that accumulated during the refolding of protein variants in which tyrosine or phenylalanine residues were individually replaced with leucine. The eight substitutions examined all caused significant changes in the intermediate distribution. In some cases, the major effect was to decrease the accumulation of intermediates containing two of the three disulfides found in the native protein, without affecting the distribution of earlier intermediates. Other substitutions, however, led to much more random distributions of the intermediates containing only one disulfide. These results indicate that the individual residues making up the hydrophobic core of the native protein make clearly distinguishable contributions to conformation and stability early in folding: The early distribution of intermediates does not appear to be determined by a general hydrophobic collapse. The effects of the substitutions were generally consistent with the structures of the major intermediates determined by NMR studies of analogs, confirming that the distribution of disulfide-bonded species is determined by stabilizing interactions within the ordered regions of the intermediates. The plasticity of the BPTI folding pathway implied by these results can be described using conformational funnels to illustrate the degree to which conformational entropy is lost at different stages in the folding of the wild-type and mutant proteins.     

Mutational analysis of the BPTI folding pathway: II. Effects of aromatic-->leucine substitutions on folding kinetics and thermodynamics.

The rates of the individual steps in the disulfide-coupled folding and unfolding of eight BPTI variants, each containing a single aromatic to leucine amino acid replacement, were measured. From this analysis, the contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide-bonded folding intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they had significantly smaller effects on the intermediates that represent the earlier stages of folding, even when the site of the substitution was located within the ordered regions of the intermediates. These results suggest that stabilizing interactions contribute less to conformational stability in the context of a partially folded intermediate than in a fully folded native protein, perhaps because of decreased cooperativity among the individual interactions. The kinetic analysis also provides new information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that these transition states are extensively unfolded. Although the substitutions caused large changes in the distribution of folding intermediates and in the rates of some steps in the folding pathway, the kinetically-preferred pathway for all of the variants involved intramolecular disulfide rearrangements, as observed previously for the wild-type protein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.     

The role of flexibility and hydration on the sequence-specific DNA recognition by the Tn916 integrase protein: a molecular dynamics analysis

The N-terminal domain of the Tn916 integrase protein (INT-DBD) is responsible for DNA binding in the process of strand cleavage and joining reactions required for transposition of the Tn916 conjugative transposon. Site-specific association is facilitated by numerous protein-DNA contacts from the face of a three-stranded beta-sheet inserted into the major groove. The protein undergoes a subtle conformational transition and is slightly unfolded in the protein-DNA complex. The conformation of many charged residues is poorly defined by NMR data but mutational studies have indicated that removal of polar side chains decreases binding affinity, while non-polar contacts are malleable. Based on analysis of the binding enthalpy and binding heat capacity, we have reasoned that dehydration of the protein-DNA interface is incomplete. This study presents results from a molecular dynamics investigation of the INT-DBD-DNA complex aimed at a more detailed understanding of the role of conformational dynamics and hydration in site-specific binding. Comparison of simulations (total of 13 ns) of the free protein and of the bound protein conformation (in isolation or DNA-bound) reveals intrinsic flexibility in certain parts of the molecule. Conformational adaptation linked to partial unfolding appears to be induced by protein-DNA contacts. The protein-DNA hydrogen-bonding network is highly dynamic. The simulation identifies protein-DNA interactions that are poorly resolved or only surmised from the NMR ensemble. Single water molecules and water clusters dynamically optimize the complementarity of polar interactions at the 'wet' protein-DNA interface. The simulation results are useful to establish a qualitative link between experimental data on individual residue's contribution to binding affinity and thermodynamic properties of INT-DBD alone and in complex with DNA.     

Change in protein flexibility upon complex formation: analysis of Ras-Raf using molecular dynamics and a molecular framework approach

Changes in flexibility upon protein-protein complex formation of H-Ras and the Ras-binding domain of C-Raf1 have been investigated using the molecular framework approach FIRST (Floppy Inclusion and Rigid Substructure Topology) and molecular dynamics simulations (MD) of in total approximately 35 ns length. In a computational time of about one second, FIRST identifies flexible and rigid regions in a single, static three-dimensional molecular framework, whose vertices represent protein atoms and whose edges represent covalent and non-covalent (hydrogen bond and hydrophobic) constraints and fixed bond angles within the protein. The two methods show a very good agreement with respect to the identification of changes in flexibility in both binding partners on a local scale. This implies that flexibility can be successfully predicted by identifying which bonds limit motion within a molecule and how they are coupled. In particular, as identified by MD, the beta-sheet in Raf shows considerably more pronounced orientational correlations in the bound state compared to the unbound state. Similarly, FIRST assigns the beta-sheet to the largest rigid cluster of the complex. Interestingly, FIRST allows us to identify that interactions across the interface (but not conformational changes upon complex formation) result in the observed rigidification. Since regions of the beta-sheet of Raf that do not interact directly with Ras become rigidified, this also demonstrates the long-range aspect to rigidity percolation. Possible implications of the change of flexibility of the Ras-binding domain of Raf on the activation of Raf upon complex formation are discussed. Finally, the sensitivity of FIRST results with respect to the representation of non-covalent interactions used as constraints is probed.     

Coarse grain lipid-protein molecular interactions and diffusion with MsbA flippase

Coarse-grained (CG) modeling has proven effective for simulating lipid bilayer dynamics on scales of biological interest. Modeling the dynamics of flexible membrane proteins within the bilayer, on the other hand, poses a considerable challenge due to the complexity of the folding or conformational landscape. In the present work, the multiscale coarse-graining method is applied to atomistic peptide-lipid "soup" simulations to develop a general set of CG protein-lipid interaction potentials. The reduced model was constructed to be compatible with recent solvent-free CG models developed for protein-protein folding and lipid-lipid model bilayer interactions. The utility of the force field was demonstrated by molecular dynamics simulation of the MsbA ABC transporter in a mixed DOPC/DOPE bilayer. An elastic network was parameterized to restrain the MsbA dimer in its open, closed and hydrolysis intermediate conformations and its impact on domain flexibility was examined. Conformational stability enabled long-time dynamics simulation of MsbA freely diffusing in a 25 nm membrane patch. Three-dimensional density analysis revealed that a shell of weakly bound "annular lipids" solvate the membrane accessible surface of MsbA and its internal substrate-binding chamber. The annular lipid binding modes, along with local perturbations in head group structure, are a function of the orientation of grooves formed between transmembrane helices and may influence the alternating access mechanism of substrate entry and translocation.     

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.     

Conformational insights on the molecular recognition processes of carbohydrate molecules by proteins and enzymes: A 3D view by using NMR

This review focuses, in a non-exhaustive manner, on the essential structural and conformational features of protein-carbohydrate interactions and on some applications of NMR spectroscopy to deal with this topic from different levels of complexity.     

Dimerization of the p53 oligomerization domain: identification of a folding nucleus by molecular dynamics simulations

Dimerization of the p53 oligomerization domain involves coupled folding and binding of monomers. To examine the dimerization, we have performed molecular dynamics (MD) simulations of dimer folding from the rate-limiting transition state ensemble (TSE). Among 799 putative transition state structures that were selected from a large ensemble of high-temperature unfolding trajectories, 129 were identified as members of the TSE via calculation of a 50% transmission coefficient from at least 20 room-temperature simulations. This study is the first to examine the refolding of a protein dimer using MD simulations in explicit water, revealing a folding nucleus for dimerization. Our atomistic simulations are consistent with experiment and offer insight that was previously unobtainable.     

Insights into the interactions between HIV-1 integrase and human LEDGF/p75 by molecular dynamics simulation and free energy calculation

Human cellular protein LEDGF/p75 (lens epithelium-derived growth factor) is an important binding partner of human immunodeficiency virus type 1 (HIV-1) integrase (IN). Without LEDGF/p75, HIV-1 can not complete its life cycle. To study the detailed interactions between LEDGF/p75 and HIV-1 IN, and then obtain the hotspots at the binding interface, 13 ns molecular dynamics simulations were carried out here. One-hundred snapshots extracted from the last 4 ns trajectories were used for calculation of binding free energy and decomposition of the energy by residue. First, the structural changes and their dynamic interactions were investigated focused on the production stage. And then, the free energy was discussed. On the basis of the above results, it could be suggested that residues Gln168, Glu170, and Thr174 in chain A of IN, Thr125, and Trp131 in chain B of IN as well as Ile365, Asp366, Phe406, and Val408 in LEDGF/p75 were responsible for their binding. These results might be helpful for discovery and design of small molecules to interrupt the interaction between HIV-1 IN and LEDGF/p75.     

Determination of potential selective inhibitors for ROCKI and ROCKII isoforms with molecular modeling techniques: structure based docking,ADMET and molecular dynamics simulation

Rho-associated protein kinases (ROCKs) are a member of the serine/threonine protein kinase family and potential therapeutic target for various diseases. This enzyme has two isoforms, Rho-associated protein kinase I (ROCKI) and Rho-associated protein kinase II (ROCKII). They share an overall 65% homology in all amino acid sequence and 92% homology in kinase domains. Since, the kinase domains of ROCKI and ROCKII are highly conserved and similar, the discovery and design of isoform-selective inhibitors are more challenging. Thus, most currently available agents that is against ROCKs exhibit low selectivity and severe side effects. Therefore, this study aimed to elucidate the interaction of compounds that indicated high potential in experimental studies against ROCKI and ROCKII enzymes in the molecular level with molecular modeling techniques. Firstly, we determined the interaction property of catalytic sites of the ROCKs by analyzing with molecular docking. Based on these results, the best ligands (50 compounds) corresponding to experimental studies were selected, and then absorption, distribution, metabolism and excretion – toxicity (ADMET) analysis of these compounds were implemented. According to these study results, the compound 40 for ROCKI and the compound 50 for ROCKII were identified as selective and highly potent inhibitors. And finally, molecular dynamics (MD) simulations were performed for the stability of ROCKs with identified compounds. In the light of this study, it will be possible to treat diseases that ROCKs have a role by developing more effective and specific ROCK inhibitors.

Communicated by Ramaswamy H. Sarma     

Toward a systems level view of the ECM and related proteins: a framework for the systematic definition and analysis of biological systems

Advances in high throughput 'omic technologies are starting to provide unprecedented insights into how components of biological systems are organized and interact. Key to exploiting these datasets is the definition of the components that comprise the system of interest. Although a variety of knowledge bases exist that capture such information, a major challenge is determining how these resources may be best utilized. Here we present a systematic curation strategy to define a systems-level view of the human extracellular matrix (ECM)--a three-dimensional meshwork of proteins and polysaccharides that impart structure and mechanical stability to tissues. Employing our curation strategy we define a set of 357 proteins that represent core components of the ECM, together with an additional 524 genes that mediate related functional roles, and construct a map of their physical interactions. Topological properties help identify modules of functionally related proteins, including those involved in cell adhesion, bone formation and blood clotting. Because of its major role in cell adhesion, proliferation and morphogenesis, defects in the ECM have been implicated in cancer, atherosclerosis, asthma, fibrosis, and arthritis. We use MeSH annotations to identify modules enriched for specific disease terms that aid to strengthen existing as well as predict novel gene-disease associations. Mapping expression and conservation data onto the network reveal modules evolved in parallel to convey tissue-specific functionality on otherwise broadly expressed units. In addition to demonstrating an effective workflow for defining biological systems, this study crystallizes our current knowledge surrounding the organization of the ECM.