ATPase and GTPase have been widely found as chemical energy-mechanical work transducers, whereas the physicochemical mechanisms are not satisfactorily understood. We addressed the problem by examining John Ross’ conjecture that...

Adenosine triphosphate (ATP) is newly expected to be involved in the clearance of amyloid beta 1-42 (A beta(42)) fibril and its precursors, A beta(42 )oligomer. Meanwhile, the microscopic mechanism of the role in dissolving the protein aggregate still remains elusive. Aiming to elucidate the mechanism, we examined effects of ATP on the conformational change and thermodynamic stability of the protomer dimer of A beta(42) pentamer and tetramer, A beta(42) (9), by employing all-atom molecular dynamics simulations. We observed interprotomer twisting and intraprotomer peeling of A beta(42) (9). These conformational changes remarkably accelerate dissociation of the protomer dimer. However, the presence of ATP itself has no positive effect on dissociation processes of the protomer dimer and a monomer from the dimer, indicating its irrelevance to decomposition of the A beta(42) oligomer. Rather, it could be supposed that ATP prevents additional binding and rebinding of A beta(42) monomers to the A beta(42) oligomer and it then converts A beta(42) oligomer into an offpathway species which is excluded from A beta(42) fibril growth processes. Interestingly, hydrophobic adenosine in ATP makes contact with A beta(42)(9) on its backbone atoms, with respect to both A beta(42) monomers on the edge of A beta(42)(9) and dissociated Afi 42 monomers in A beta(42)(9). These roles of ATP would be applied without regard to the structural polymorphism of the A beta(42) fibril.

Thrombin is a serine protease involved in the blood coagulation reaction, and it shows maximum enzymatic activity in the presence of Na+ It has been supposed that Na+ binding promotes conversion from the inactive form, with a collapsed primary substrate pocket (S1 pocket), to the active form, with a properly formed S1 pocket. However, the evidence supporting this activation mechanism was derived from the X-ray crystallographic structures solved under nonphysiological conditions and using thrombin mutants; thus, it still remains elusive whether the activation mechanism is actually attributed to Na+ binding. To address the problem, we employed all-atom molecular dynamics simulations for both active and inactive forms of thrombin in the presence and absence of Na+ binding and examined the effect of Na+ binding on S1-pocket formation. In contrast to the conventional supposition, we revealed that Na+ binding does not prevent S1-pocket collapse virtually, but rather, the bound Na+ can move to the S1 pocket, thus blocking substrate access directly. Additionally, it was clarified that Na+ binding does not promote S1-pocket formation. According to these insights, we concluded that Na+ binding is irrelevant to the interconversion between the inactive and active forms of thrombin.

Thrombin has been studied as a paradigmatic protein of Na+- activated allosteric enzymes. Earlier structural studies suggest that Na+-binding promotes the thrombin-substrate association reaction. However, it is still elusive because (1) the structural change, driven by Na+-binding, is as small as the thermal fluctuation, and (2) the bound Na+ is close to Asp189 in the primary substrate binding pocket (S1-pocket), possibly preventing substrate access via repulsive interaction. It still remains a matter of debate whether Na+-binding actually promotes the reaction. To solve this problem, we examined the effect of Na+ on the reaction by employing molecular dynamics (MD) simulations. By executing independent 210 MD simulations of apo and holo systems, we obtained 80 and 26 trajectories undergoing substrate access to S1-pocket, respectively. Interestingly, Na+-binding results in a 3-fold reduction of the substrate access. Furthermore, we examined works for the substrate access and release, and found that Na+-binding is disadvantageous for the presence of the substrate in the S1-pocket. These observations provide the insight that the bound Na+ is essentially a negative effecter in thrombin-substrate stereospecific complex formation. The insight rationalizes an enigmatic feature of thrombin, relatively low Na+-binding affinity. This is essential to reduce the disadvantage of Na+-binding in the substrate-binding.

Upon protein-substrate association reaction, dewetting of the substrate-binding pocket is one of the rate-limiting processes. However, understanding the microscopic mechanism still remains challenging because of practical limitations of experimental methodologies. We have addressed the problem here by using molecular dynamics (MD) simulation of the thrombin-substrate association reaction. During the MD simulation, ArgP1 in a substrate accessed thrombin's substrate-binding pocket and formed specific hydrogen bonds (H-bonds) with Asp189 in thrombin, while the catalytic serine of thrombin was still away from the substrate's active site. It is assumed that the thrombin-substrate association reaction is regulated by a stepwise mechanism. Furthermore, in the earlier stage of ArgP1 access to the pocket, we observed that ArgP1 was spatially separated from Asp189 by two water molecules in the pocket. These water molecules transferred from the pocket, followed by the specific H-bond formation between thrombin and the substrate. Interestingly, they were not evacuated directly from the pocket to the bulk solvent, but moved to the water channel of thrombin. This observation indicates that the channel plays functional roles in dewetting upon the association reaction.

We shed light on important roles of unbound Na+ molecules in enzymatic activation of thrombin. Molecular mechanism of Na+-activation of thrombin has been discussed in the context of allostery. However, the recent challenge to redesign K+-activated thrombin revealed that the allosteric interaction is insufficient to explain the mechanism. Under these circumstances, we have examined the roles of unbound Na+ molecule in maximization of thrombin-substrate association reaction rate. We performed all-atomic molecular dynamics (MD) simulations of thrombin in the presence of three different cations; Li+, Na+, and Cs+. Although these cations are commonly observed in the vicinity of the S1-pocket of thrombin, smaller cations are distributed more densely and extensively than larger ones. This suggests the two observation rules: (i) thrombin surrounded by Na+ is at an advantage in the initial step of association reaction, namely, the formation of an encounter complex ensemble, and (ii) the presence of Na+ molecules does not necessarily have an advantage in the final step of association reaction, namely, the formation of the stereospecific complex. In conclusion, we propose a conjecture that unbound Na+ molecules also affect the maximization of rate constant of thrombin-substrate association reaction through optimally forming an encounter complex ensemble.

In this study, we demonstrate that U1A-RNA molecular recognition is mediated by a combined mechanism of conformational selection and induced fit. The binding of U1A to RNA has been discussed in the context of induced fit that involves the reorientation of the alpha-helix in the C-terminal region (Helix-C) of U1A to permit RNA access only when U1A correctly recognizes RNA. However, according to our molecular dynamics simulations, even in the absence of RNA, Helix-C spontaneously reoriented to permit RNA access. Nonetheless, such a conformational change was still incomplete. Helix-C was often partially or even fully unfolded and in an infrequent RNA-accessible conformation, which can be detected using state-of-the-art nuclear magnetic resonance methodology. These results suggest that the formation of an energetically stabilized complex is promoted by specific interactions between U1A and RNA. In conclusion, in the recognition of RNA by U1A protein, we propose a combined mechanism that requires the reorientation of Helix-C and the subsequent contact with RNA through conformational selection, although the stabilization of the U1A RNA complex is caused by induced fit. We further propose a modification to the conventional assumption regarding the mechanism of U1A RNA molecular recognition.

Protein allostery is essential for vital activities. Allosteric regulation of human hemoglobin (HbA) with two quaternary states T and R has been a paradigm of allosteric structural regulation of proteins. It is widely accepted that oxygen molecules (O-2) act as a "site-specific'' homotropic effector, or the successive O-2 binding to the heme brings about the quaternary regulation. However, here we show that the site-specific allosteric effect is not necessarily only a unique mechanism of O-2 allostery. Our simulation results revealed that the solution environment of high O-2 partial pressure enhances the quaternary change from T to R without binding to the heme, suggesting an additional "non-site-specific'' allosteric effect of O-2. The latter effect should play a complementary role in the quaternary change by affecting the intersubunit contacts. This analysis must become a milestone in comprehensive understanding of the allosteric regulation of HbA from the molecular point of view.

The fragment molecular orbital (FMO) method can calculate the electronic structure of macromolecules such as DNA by dividing them into several fragments and introducing suitable approximations. To establish guiding principles for FMO calculation of DNA, benchmark tests were performed for several small DNA models consisting of one or two bases or two base pairs. The effects of several factors on the accuracy of FMO calculations were investigated, including the methods used to fragment the nucleotide units, approximations for the electrostatic potential, charge neutralization, and electron correlation. It was found that charge neutralization is indispensable for the reliable calculation of energies and spatial distribution of molecular orbitals, but not necessarily so for inter-fragment interaction energy analyses, such as calculation of the base-base interaction. The electrostatic approximations were shown to have only an insignificant effect on the qualitative nature of the calculations. It was also confirmed that the base-base stacking energy can be reproduced semi-quantitatively by the Moller-Plesset second-order perturbation (MP2) method though with some overestimation, and that the overestimation can be alleviated by the spin-component-scaled MP2 method. (C) 2014 Elsevier B.V. All rights

The heme oxygen (O-2) binding site of human hemoglobin (HbA) is buried in the interior of the protein, and there is a debate over the O-2 entry pathways from solvent to the binding site. As a first step to understand HbA O-2 binding process at the atomic level, we detected all significant multiple O-2 entry pathways from solvent to the binding site in the alpha and beta subunits of the T-state tetramer HbA by utilizing ensemble molecular dynamics (MD) simulation. By executing 128 independent 8 ns MD trajectories in O-2-rich aqueous solvent, we simulated the O-2 entry processes and obtained 141 and 425 O-2 entry events in the alpha and beta subunits of HbA, respectively. We developed the intrinsic pathway identification by clustering method to achieve a persuasive visualization of the multiple entry pathways including both the shapes and relative importance of each pathway. The rate constants of O-2 entry estimated from the MD simulations correspond to the experimentally observed values, suggesting that O-2 ligands enter the binding site through multiple pathways. The obtained multiple pathway map can be utilized for future detailed analysis of HbA O-2 binding process.

Effective interactions between amino acid residues in antigen-antibody complex of influenza virus hemagglutinin (HA) protein can be evaluated in terms of the inter-fragment interaction energy (IFIE) analysis with the fragment molecular orbital (FMO) method, in which each fragment contains the side chain of corresponding amino acid residue. We have carried out the FMO-MP2 (second-order Moeller-Plesset) calculation for the complex of HA antigen and Fab antibody of influenza virus H3N2 A/Aichi/2/68 and obtained the IFIE values between each amino acid residue in HA and the whole antibody as the sums over the residues contained in the latter. Combining this IFIE data with experimental data for hemadsorption activity of HA mutants, we succeeded in theoretically explaining the mutations in HA observed after the emergence of influenza virus H3N2 A/Aichi/2/68 in an earlier study, except for those of THR83. In the present study, we employ an alternative way of fragment division in the FMO calculation at the carbonyl C site of the peptide bond instead of the Cα site used in the previous work, which provides revised IFIE values consistent with all the historical mutation data in the antigenic region E of HA including the case of THR83 in the present prediction scheme for probable mutations in HA. © 2011 Springer-Verlag.

Mobility change of water molecules in ionic solutions has been a long standing issue in the field of spectroscopy; however, the molecular mechanism is still controversial. To address this issue, molecular dynamics (MD) simulations are considered to be useful tools because MD simulations can provide deeper insights into dynamics of water molecules at the atomic level, compensating for the limitation of experimental observations. However, the reliability of these simulations is significant and strongly dependent on the molecular models employed. We examined the reproduction of the dynamics of water molecules under the influence of alkali and halide monovalent ions by using the framework of classical MD simulations. Conventional water models were combined with non-polarizable ion models. The decreased mobility of water molecules was reproduced in our simulations while the increased mobility could not be reproduced. However, from the examined water models, the TIP5P model can be promising to reproduce the experimental results if ion water interactions are improved. (C) 2011 Elsevier B.V. All rights reserved.

We propose a novel concept associated with the relationship between structure and function in biomolecular systems. We performed a 75 nanoseconds molecular dynamics (MD) simulation for an RNA-binding protein, neurooncological ventral antigen (NOVA), and examined its physico-chemical properties. NOVA dissociated from the NOVA-RNA complex showed a large conformational change: formation of intra-molecular hydrogen bonds between the C-terminal region and the loop structure located at the middle of amino acid sequence. The free energy analysis suggests that the deformed structure is more stabilized in macromolecular crowding environment where the dielectric constant is smaller than 5. The solvent accessible surface area (SASA) analysis indicates that NOVA enhances the efficiency of association with RNA by changing the relative SASA for the target sequence in RNA molecules. Based on the obtained results, we propose a novel concept of spontaneous adjustment mechanism to explain the structural and energetic changes observed for NOVA in the free state.

We report the molecular mechanism of protein-RNA complex stabilization based on the electronic state calculation Fragment molecular orbital (FMO) method based quantum mechanical calculations were performed for neuro-oncological ventral antigen (NOVA)-RNA complex system The inter-molecular interactions and their effects on the electronic state of NOVA were examined in the framework of ab initio quantum calculation The strength of inter molecular interactions was evaluated using inter-fragment interaction energies (IFIEs) associated with residue-RNA base and residue-RNA backbone interactions Under the influence of Inter-molecular interactions the change of electronic state of NOVA upon the complex formation was examined based on IFIE values associated with ultra-NOVA residue-residue interactions and the change of atomic charges by each residue The results indicated that non-specifically recognized bases contributed to the stability of the complex as well as specifically recognized bases and that the secondary structure of NOVA was remarkably associated with the change of electronic state upon the complex formation (C) 2010 Elsevier B V All rights reserved

The RNA-binding proteins (RBPs) can recognize and bind their target RNAs specifically according to RNA sequences and/or 3D structures, and can thus express their functions. One of the RBPs, Pumilio, has an RNA-binding domain, Puf domain, in the C-terminal region, which recognizes the RNA sequence (Figure 1) and the 2' hydroxyl group in ribose. The Puf domain consists of 8 tandem modules (Figure 2) and forms the complexes with RNA containing specific sequences (Figure 3). Further, RNA base mutational experiments suggest that each base in the sequence differs in the contribution to the binding free energy of Puf domain-RNA complex formation. In this study, we employ molecular dynamics (MD) simulation to quantitatively estimate the contribution of each base and amino acid to complex stabilization. We analyze the MD trajectory of Puf domain-RNA complex system and calculate the frequencies of hydrogen bond formation at the Puf domain-RNA interface (Figures 6, 7). We also calculate the enthalpy term of the binding free energy of Puf domain-RNA complex, decompose it into enthalpy per residue (Figure 8), and compare the contributions of each amino acid and RNA base. From these analyses, we conclude that each base does not uniquely contribute to the stabilization of the complex and that amino acid residues around the binding interface are key factors of stabilization in Puf domain.

A visualization method for inter-fragment interaction energies (IFIEs) of biopolymers is presented on the basis of the fragment molecular orbital (FMO) method. The IFIEs appropriately illustrate the information about the interaction energies between the fragments consisting of amino acids, nucleotides and other molecules. The IFTEs are usually analyzed in a matrix form called an IFTE matrix. Analyzing the IFIE matrix, we detect important fragments for the function of biomolecular systems and quantify the strength of interaction energies based on quantum chemistry, including the effects of charge transfer, electronic polarization and dispersion force. In this study, by analyzing a protein-DNA complex, we report a visual representation of the IFTE matrix, a so-called IFTE map. We comprehensively examine what information the THE map contains concerning structures and stabilities of the protein-DNA complex. (c) 2007 Elsevier B.V. All rights reserved.

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.