A Free-Energy Landscape Analysis of Calmodulin Obtained from an NMR Data-Utilized Multi-Scale Divide-and-Conquer Molecular Dynamics Simulation.

Calmodulin (CaM) is a multifunctional calcium-binding protein, which regulates a variety of biochemical processes. CaM acts through its conformational changes and complex formation with its target enzymes. CaM consists of two globular domains (N-lobe and C-lobe) linked by an extended linker region. Upon calcium binding, the N-lobe and C-lobe undergo local conformational changes, followed by a major conformational change of the entire CaM to wrap the target enzyme. However, the regulation mechanisms, such as allosteric interactions, which regulate the large structural changes, are still unclear. In order to investigate the series of structural changes, the free-energy landscape of CaM was obtained by multi-scale divide-and-conquer molecular dynamics (MSDC-MD). The resultant free-energy landscape (FEL) shows that the Ca2+ bound CaM (holo-CaM) would take an experimentally famous elongated structure, which can be formed in the early stage of structural change, by breaking the inter-domain interactions. The FEL also shows that important interactions complete the structural change from the elongated structure to the ring-like structure. In addition, the FEL might give a guiding principle to predict mutational sites in CaM. In this study, it was demonstrated that the movement process of macroscopic variables on the FEL may be diffusive to some extent, and then, the MSDC-MD is suitable to the parallel computation.

Experimental and theoretical study on converting myoglobin into a stable domain-swapped dimer by utilizing a tight hydrogen bond network at the hinge region.

Various factors, such as helical propensity and hydrogen bonds, control protein structures. A frequently used model protein, myoglobin (Mb), can perform 3D domain swapping, in which the loop at the hinge region is converted to a helical structure in the dimer. We have previously succeeded in obtaining monomer-dimer equilibrium in the native state by introducing a high α-helical propensity residue, Ala, to the hinge region. In this study, we focused on another factor that governs the protein structure, hydrogen bonding. X-ray crystal structures and thermodynamic studies showed that the myoglobin dimer was stabilized over the monomer when keeping His82 to interact with Lys79 and Asp141 through water moleclues and mutating Leu137, which was located close to the H-bond network at the dimer hinge region, to a hydrophilic amino acid (Glu or Asp). Molecular dynamics simulation studies confirmed that the number of H-bonds increased and the α-helices at the hinge region became more rigid for mutants with a tighter H-bond network, supporting the hypothesis that the myoglobin dimer is stabilized when the H-bond network at the hinge region is enhanced. This demonstrates the importance and utility of hydrogen bonds for designing a protein dimer from its monomer with 3D domain swapping.

Atomistic detailed free-energy landscape of intrinsically disordered protein studied by multi-scale divide-and-conquer molecular dynamics simulation.

Calcineurin (CaN) is a eukaryotic serine/threonine protein phosphatase activated by both Ca2+ and calmodulin (CaM), including intrinsically disordered region (IDR). The region undergoes folding into an α-helix form in the presence Ca2+ -loaded CaM. To sample the ordered structure of the IDR by conventional all atom model (AAM) molecular dynamics (MD) simulation, the IDR and Ca2+ -loaded CaM must be simultaneously treated. However, it is time-consuming task because the coupled folding and binding should include repeated binding and dissociation. Then, in this study, we propose novel multi-scale divide-and-conquer MD (MSDC-MD), which combines AAM-MD and coarse-grained model MD (CGM-MD). To speed up the conformation sampling, MSDC-MD simulation first treats the IDR by CGM to sample conformations from wide conformation space; then, multiple AAM-MD in a limited area is initiated using the resultant CGM conformation, which is reconstructed by homology modeling method. To investigate performance, we sampled the ordered conformation of the IDR using MSDC-MD; the root-mean-square distance (RMSD) with respect to the experimental structure was 2.23 Å.

A structural comparison of 'real' and 'model' calmodulin clarified allosteric interactions regulating domain motion.

Calmodulin (CaM) is a multifunctional calcium-binding protein, which regulates various biochemical processes. CaM acts via structural changes and complex forming with its target enzymes. CaM has two globular domains (N-lobe and C-lobe) connected by a long linker region. Upon calcium binding, the N-lobe and C-lobe undergo local conformational changes, after that, entire CaM wraps the target enzyme through a large conformational change. However, the regulation mechanism, such as allosteric interactions regulating the conformational changes, is still unclear. In order to clarify the allosteric interactions, in this study, experimentally obtained 'real' structures are compared to 'model' structures lacking the allosteric interactions. As the allosteric interactions would be absent in calcium-free CaM (apo-CaM), allostery-eliminated calcium-bound CaM (holo-CaM) models were constructed by combining the apo-CaM's linker and the holo-CaM's N- and C-lobe. Before the comparison, the 'real' and 'model' structures were clustered and cluster-cluster relationship was determined by a principal component analysis. The structures were compared based on the relationship, then, a distance map and a contact probability analysis clarified that the inter-domain motion is regulated by several groups of inter-domain contacting residue pairs. The analyses suggested that these residues cause inter-domain translation and rotation, and as a consequence, the motion encourage structural diversity. The resultant diversity would contribute to the functional versatility of CaM.

Residue-residue interactions regulating the Ca2+-induced EF-hand conformation changes in calmodulin.

Calmodulin (CaM) is a Ca2+-binding messenger protein having four Ca2+-binding motifs named 'EF-hand'; the EF-hand motifs undergo a conformation change induced by Ca2+-binding. In order to study how Ca2+-binding induces the conformation change of EF-hand motifs and which residues are involved in the reaction, two 1µ second long MD simulations were independently performed from the apo- and holo-CaM and their structures and interactions were compared. The Ca2+-binding weakens the helix-helix interaction in all EF-hand, however, the holo-CaM MD adopted the close-like form. The correlation coefficients obtained from the two MDs show the residues comprising interactions being involved in their close-open conformation changes; most of these residues are hydrophobic amino acids but some of them are hydrophilic (T34, H107, N111 and Q143). The hydrophilic residues are expected to lock the EF-hands by their side-chains and main-chain carbonyl oxygen of another hydrophobic residue. Furthermore, the interaction pattern of EF-hand3 and 4 are similar to each other. On the other hand, the interaction pattern of EF-hand2 is different from others; its polar residues are expected to play an important role in regulating the EF-hand2 conformation.

Quality assessment methods for 3D protein structure models based on a residue-residue distance matrix prediction.

In the absence of experimentally determined three dimensional (3D) structures of proteins, the prediction of protein structures using computational methods is a standard alternative approach in bioinformatics. When using the predicted protein models to compute the native structure of an unknown target protein, estimating the actual quality of the protein models is important for selecting the best or near-best model. Moreover, estimates of the differences between the protein models and the native protein structure are obviously useful to end users who can then decide on the utility of the models for their specific problems. This article describes two new single-model quality assessment (QA) programs, pure single-model QA method (psQA) and a template based QA method (tbQA), that we developed. psQA is a pure single-model QA program that uses a neural network method to predict residue-residue distance matrices of the native protein structures. tbQA is a quasi-single-model QA program that mainly uses target-template sequence alignments and template structures. The performance of these two model QA programs was analyzed in a data set of 24022 models for 94 targets from the 10th critical assessment of protein structure prediction (CASP10) experiment.

Community-wide evaluation of methods for predicting the effect of mutations on protein-protein interactions.

Community-wide blind prediction experiments such as CAPRI and CASP provide an objective measure of the current state of predictive methodology. Here we describe a community-wide assessment of methods to predict the effects of mutations on protein-protein interactions. Twenty-two groups predicted the effects of comprehensive saturation mutagenesis for two designed influenza hemagglutinin binders and the results were compared with experimental yeast display enrichment data obtained using deep sequencing. The most successful methods explicitly considered the effects of mutation on monomer stability in addition to binding affinity, carried out explicit side-chain sampling and backbone relaxation, evaluated packing, electrostatic, and solvation effects, and correctly identified around a third of the beneficial mutations. Much room for improvement remains for even the best techniques, and large-scale fitness landscapes should continue to provide an excellent test bed for continued evaluation of both existing and new prediction methodologies.

Simple and effective application of the Wang-Landau method for multicanonical molecular dynamics simulation.

We propose a novel application of the Wang-Landau method (WLM) for multicanonical molecular dynamics (McMD) simulations. Originally, WLM was developed for Monte Carlo (MC) simulations. Fundamentally, WLM remarkably reduces simulation efforts because it estimates the optimal multicanonical energy function automatically. When WLM is applied to McMD, not only the multicanonical energy but also energy gradient must be estimated adequately. However, because of the rugged multicanonical energy function at the early simulation stage, applications of WLM for MD simulations are difficult and require a smoothing procedure: simulation efforts such as cubic-spline extrapolation and gathering multiple preruns are utilized for smoothing. We propose a simple and effective smoothing method that requires only one additional equation and two time-dependent parameters. As a result, our method produced the correct multicanonical energy function and succeeded in the flat sampling of a small biomolecule with reduced simulation effort.

Free Energy Landscapes of Alanine Dipeptide in Explicit Water Reproduced by the Force-Switching Wolf Method.

Precise and rapid calculation of long-range interactions is of crucial importance for molecular dynamics (MD) and Monte Carlo simulations. Instead of the Ewald method or its high speed variant, PME, we applied our novel method, called the force-switching Wolf method, to computation of the free energy landscapes of a short peptide in explicit water. Wolf and co-workers showed that long-range electrostatic energy under a periodic boundary condition can be well reproduced even by truncating the contribution from the distant charges, when the charge neutrality is taken into account. We recently applied the procedure proposed by Wolf and co-workers to a mathematically consistent MD theory by means of a force-switching scheme, and we show that the total electrostatic energy for sodium chloride liquid was well conserved and stable during the MD simulation with the force-switching Wolf method. Our current results for an aqueous peptide solution with a series of canonical and multicanonical molecular dynamics simulations show that the force-switching Wolf method is not only in good accordance with the energies and forces calculated by the conventional PME method but also properly reproduces the solvation and the free energy landscapes of the peptide at 300 K.

Enhanced free-energy calculation using multiscale simulation.

We propose a multiscale simulation method combining the efficiency of a coarse-grained model (CGM) and the accuracy of an all-atom model (AAM) for free-energy landscape calculation of protein systems. A protein's conformation space is quickly searched first using CGM. Then the obtained information is incorporated into AAM simulations. The free-energy landscape is subsequently obtained from AAM simulations. This method was tested on chignolin folding. The results demonstrated that the computational time was reduced by as much as 90%.