Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum.

The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.

Fragment Molecular Orbital Based Interaction Analyses on COVID-19 Main Protease - Inhibitor N3 Complex (PDB ID: 6LU7).

The worldwide spread of COVID-19 (new coronavirus found in 2019) is an emergent issue to be tackled. In fact, a great amount of works in various fields have been made in a rather short period. Here, we report a fragment molecular orbital (FMO) based interaction analysis on a complex between the SARS-CoV-2 main protease (Mpro) and its peptide-like inhibitor N3 (PDB ID: 6LU7). The target inhibitor molecule was segmented into five fragments in order to capture site specific interactions with amino acid residues of the protease. The interaction energies were decomposed into several contributions, and then the characteristics of hydrogen bonding and dispersion stabilization were made clear. Furthermore, the hydration effect was incorporated by the Poisson-Boltzmann (PB) scheme. From the present FMO study, His41, His163, His164, and Glu166 were found to be the most important amino acid residues of Mpro in interacting with the inhibitor, mainly due to hydrogen bonding. A guideline for optimizations of the inhibitor molecule was suggested as well based on the FMO analysis.

Cm3+/Eu3+ induced structural, mechanistic and functional implications for calmodulin.

Trivalent actinides and their lanthanide homologues are being scrutinized for their potential health risk when ingested as a result of a range of industrial activities such as mining. Importantly, these ions are known to exhibit high affinity towards calmodulin (CaM). In case of their inadvertent uptake, the holoproteins that are occupied by these cations may block signal transduction pathways or increase the concentration of these ions in intact cells, which could lead to accumulation in human organs. Accordingly, this investigation employed spectroscopy, computational chemistry, calorimetry, and biochemistry to study the results of metal ion substitution on the protein structure, enzymatic activity and chemo- and cytotoxicity of An3+/Ln3+ ions. As will be demonstrated herein, our data confirm the higher affinity of Cm3+ and Eu3+ compared to Ca2+ to all 4 binding sites of CaM, with one site differing from the remaining three. This higher-affinity site will complex Eu3+ in an exothermic fashion; in contrast, ion binding to the three lower-affinity EF-hands was found to be endothermic. The overall endothermic binding process is ascribed to the loss of the hydration shells of the trivalent ions upon protein binding. These findings are supported by extensive quantum chemical calculations of full holo-CaM, which were performed at the MP2 level using the fragment molecular orbital method. The exceptional binding site (EF-hand 3) features fewer negatively charged residues compared to the other EF-hands, thereby allowing Eu3+ and Cm3+ to carry one or two additional waters compared to Ca2+-CaM, while also causing the structure of Cm3+/Eu3+-CaM to become slightly disordered. Moreover, the enzymatic activity decreases somewhat in comparison to Ca2+-CaM. By utilizing a combination of techniques, we were able to generate a comprehensive picture of the CaM-actinide/lanthanide system from the molecular level to its functional impact. Such knowledge could also be applied to other metal-binding proteins.

Potentiating Antigen-Specific Antibody Production with Peptides Obtained from In Silico Screening for High-Affinity against MHC-II.

Monoclonal antibodies with high affinity and specificity are essential for research and clinical purposes, yet remain difficult to produce. Agretope peptides that can potentiate antigen-specific antibody production have been reported recently. Here, we screened in silico for peptides with higher affinity against the agretope binding pocket in the MHC-II. The screening was based on the 3D crystal structure of a complex between MHC-II and a 14-mer peptide consisting of ovalbumin residues 323-339. Using this 14-mer peptide as template, we constructed a library of candidate peptides and screened for those that bound tightly to MHC-II. Peptide sequences that exhibited a higher binding affinity than the original ovalbumin peptide were identified. The peptide with the highest binding affinity was synthesized and its ability to boost antigen-specific antibody production in vivo and in vitro was assessed. In both cases, antigen-specific IgG antibody production was potentiated. Monoclonal antibodies were established by in vitro immunization using this peptide as immunostimulant, confirming the usefulness of such screened peptides for monoclonal antibody production.

Electron-correlated fragment-molecular-orbital calculations for biomolecular and nano systems.

Recent developments in the fragment molecular orbital (FMO) method for theoretical formulation, implementation, and application to nano and biomolecular systems are reviewed. The FMO method has enabled ab initio quantum-mechanical calculations for large molecular systems such as protein-ligand complexes at a reasonable computational cost in a parallelized way. There have been a wealth of application outcomes from the FMO method in the fields of biochemistry, medicinal chemistry and nanotechnology, in which the electron correlation effects play vital roles. With the aid of the advances in high-performance computing, the FMO method promises larger, faster, and more accurate simulations of biomolecular and related systems, including the descriptions of dynamical behaviors in solvent environments. The current status and future prospects of the FMO scheme are addressed in these contexts.

Prediction of Binding Pose and Affinity of Nelfinavir, a SARS-CoV-2 Main Protease Repositioned Drug, by Combining Docking, Molecular Dynamics, and Fragment Molecular Orbital Calculations.

A novel in silico drug design procedure is described targeting the Main protease (Mpro) of the SARS-CoV-2 virus. The procedure combines molecular docking, molecular dynamics (MD), and fragment molecular orbital (FMO) calculations. The binding structure and properties of Mpro were predicted for Nelfinavir (NFV), which had been identified as a candidate compound through drug repositioning, targeting Mpro. Several poses of the Mpro and NFV complexes were generated by docking, from which four docking poses were selected by scoring with FMO energy. Then, each pose was subjected to MD simulation, 100 snapshot structures were sampled from each of the generated MD trajectories, and the structures were evaluated by FMO calculations to rank the pose based on binding energy. Several residues were found to be important in ligand recognition, including Glu47, Asp48, Glu166, Asp187, and Gln189, all of which interacted strongly with NFV. Asn142 is presumably regarded to form hydrogen bonds or CH/π interaction with NFV; however, in the present calculation, their interactions were transient. Moreover, the tert-butyl group of NFV had no interaction with Mpro. Identifying such strong and weak interactions provides candidates for maintaining and substituting ligand functional groups and important suggestions for drug discovery using drug repositioning. Besides the interaction between NFV and the amino acid residues of Mpro, the desolvation effect of the binding pocket also affected the ranking order. A similar procedure of drug design was applied to Lopinavir, and the calculated interaction energy and experimental inhibitory activity value trends were consistent. Our approach provides a new guideline for structure-based drug design starting from a candidate compound whose complex crystal structure has not been obtained.

FMO-MD simulations on the hydration of formaldehyde in water solution with constraint dynamics.

Full-quantum mechanical fragment molecular orbital-based molecular dynamics (FMO-MD) simulations were applied to the hydration reaction of formaldehyde in water solution under neutral conditions. Two mechanisms, a concerted and a stepwise one, were considered with respect to the nucleophilic addition and the proton transfer. Preliminary molecular orbital calculations by means of polarized continuum model reaction field predicted that the hydration prefers a concerted mechanism. Because the calculated activation barriers were too high for free FMO-MD simulations to give reactive trajectories spontaneously, a More O'Ferrall-Jencks-type diagram was constructed from the statistical analysis of the FMO-MD simulations with constraint dynamics. The diagram showed that the hydration proceeds through a zwitterionic-like (ZW-like) structure. The free energy changes along the reaction coordinate calculated by means of the blue moon ensemble for the hydration and the amination of formaldehyde indicated that the hydration proceeds through a concerted process through the ZW-like structure, whereas the amination goes through a stepwise mechanism with a ZW intermediate. In inspection of the FMO-MD trajectories, water-mediated cyclic proton transfers were observed in both reactions on the way from the ZW-like structure to the product. These proton transfers also have an asynchronous character, in which deprotonation from the nucleophilic oxygen atom (or nitrogen atom for amination) precedes the protonation of the carbonyl oxygen atom. The results showed the strong advantage of the FMO-MD simulations to obtain detailed information at a molecular level for solution reactions.

Interaction analyses of SARS-CoV-2 spike protein based on fragment molecular orbital calculations.

At the stage of SARS-CoV-2 infection in human cells, the spike protein consisting of three chains, A, B, and C, with a total of 3300 residues plays a key role, and thus its structural properties and the binding nature of receptor proteins to host human cells or neutralizing antibodies has attracted considerable interest. Here, we report on interaction analyses of the spike protein in both closed (PDB-ID: 6VXX) and open (6VYB) structures, based on large-scale fragment molecular orbital (FMO) calculations at the level of up to the fourth-order Møller-Plesset perturbation with singles, doubles, and quadruples (MP4(SDQ)). Inter-chain interaction energies were evaluated for both structures, and a mutual comparison indicated considerable losses of stabilization energies in the open structure, especially in the receptor binding domain (RBD) of chain-B. The role of charged residues in inter-chain interactions was illuminated as well. By two separate calculations for the RBD complexes with angiotensin-converting enzyme 2 (ACE2) (6M0J) and B38 Fab antibody (7BZ5), it was found that the binding with ACE2 or antibody partially compensated for this stabilization loss of RBD.

Dynamic Cooperativity of Ligand-Residue Interactions Evaluated with the Fragment Molecular Orbital Method.

By the splendid advance in computation power realized with the Fugaku supercomputer, it has become possible to perform ab initio fragment molecular orbital (FMO) calculations for thousands of dynamic structures of protein-ligand complexes in a parallel way. We thus carried out electron-correlated FMO calculations for a complex of the 3C-like (3CL) main protease (Mpro) of the new coronavirus (SARS-CoV-2) and its inhibitor N3 incorporating the structural fluctuations sampled by classical molecular dynamics (MD) simulation in hydrated conditions. Along with a statistical evaluation of the interfragment interaction energies (IFIEs) between the N3 ligand and the surrounding amino-acid residues for 1000 dynamic structure samples, in this study we applied a novel approach based on principal component analysis (PCA) and singular value decomposition (SVD) to the analysis of IFIE data in order to extract the dynamically cooperative interactions between the ligand and the residues. We found that the relative importance of each residue is modified via the structural fluctuations and that the ligand is bound in the pharmacophore in a dynamic manner through collective interactions formed by multiple residues, thus providing new insight into structure-based drug discovery.

Taking Water into Account with the Fragment Molecular Orbital Method.

This chapter describes the current status of development of the fragment molecular orbital (FMO) method for analyzing the electronic state and intermolecular interactions of biomolecular systems in solvent. The orbital energies and the inter-fragment interaction energies (IFIEs) for a specific molecular structure can be obtained directly by performing FMO calculations by exposing water molecules and counterions around biomolecular systems. Then, it is necessary to pay attention to the thickness of the water shell surrounding the biomolecules. The single-point calculation for snapshots from MD trajectory does not incorporate the effects of temperature and configurational fluctuation, but the SCIFIE (statistically corrected IFIE) method is proposed as a many-body correlated method that partially compensates for this deficiency. Furthermore, implicit continuous dielectric models have been developed as effective approaches to incorporating the screening effect of the solvent in thermal equilibrium, and we illustrate their usefulness for theoretical evaluation of IFIEs and ligand-binding free energy on the basis of the FMO-PBSA (Poisson-Boltzmann surface area) method and other computational methods.

Fragment Molecular Orbital method-based Molecular Dynamics (FMO-MD) as a simulator for chemical reactions in explicit solvation.

Fragment Molecular Orbital based-Molecular Dynamics (FMO-MD, Komeiji et al., Chem Phys Lett 2003, 372, 342) is an ab initio MD method suitable for large molecular systems. Here, FMO-MD was implemented to conduct full quantum simulations of chemical reactions in explicit solvation. Several FMO-MD simulations were performed for a sphere of water to find a suitable simulation protocol. It was found that annealing of the initial configuration by a classical MD brought the subsequent FMO-MD trajectory to faster stabilization, and also that use of bond constraint in the FMO-MD heating stage effectively reduced the computation time. Then, the blue moon ensemble method (Sprik and Ciccotti, J Chem Phys 1998, 109, 7737) was implemented and was tested by calculating free energy profiles of the Menschutkin reaction (H3N + CH3Cl --> +H3NCH3 + Cl-) in the presence and absence of the solvent water via FMO-MD. The obtained free energy profiles were consistent with the Hammond postulate in that stabilization of the product by the solvent, namely hydration of Cl-, shifted the transition state to the reactant-side. Based on these FMO-MD results, plans for further improvement of the method are discussed.

Water-mediated interactions in the CRP-cAMP-DNA complex: does water mediate sequence-specific binding at the DNA primary-kink site?

The cyclic AMP receptor protein (CRP) of Escherichia coli binds preferentially to DNA sequences possessing a T:A base pair at position 6 (at which the DNA becomes kinked), but with which it does not form any direct interactions. It has been proposed that indirect readout is involved in CRP-DNA binding, in which specificity for this base pair is primarily related to sequence effects on the energetic susceptibility of the DNA to kink formation. In the current study, the possibility of contributions to indirect readout by water-mediated hydrogen bonding of CRP with the T:A base pair was investigated. A 1.0 ns molecular dynamics simulation of the CRP-cAMP-DNA complex in explicit solvent was performed, and assessed for water-mediated CRP-DNA hydrogen bonds; results were compared to several X-ray crystal structures of comparable complexes. While several water-mediated CRP-DNA hydrogen bonds were identified, none of these involved the T:A base pair at position 6. Therefore, the sequence specificity for this base pair is not likely enhanced by water-mediated hydrogen bonding with the CRP.

Visualization analysis of inter-fragment interaction energies of CRP-cAMP-DNA complex based on the fragment molecular orbital method.

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 IFIEs are usually analyzed in a matrix form called an IFIE 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 IFIE matrix, a so-called IFIE map. We comprehensively examine what information the IFIE map contains concerning structures and stabilities of the protein-DNA complex.

Hydration of ligands of influenza virus neuraminidase studied by the fragment molecular orbital method.

The fragment molecular orbital (FMO) method was applied to quantum chemical calculations of neuramic acid, the natural substrate of the influenza virus neuraminidase, and two of its competitive inhibitors, Oseltamivir (Tamiful(®)) and Zanamivir (Relenza(®)), to investigate their hydrated structures and energetics. Each of the three ligands was immersed in an explicit water solvent, geometry-optimized by classical MM and QM/MM methods, and subjected to FMO calculations with 2-, 3-, and 4-body corrections under several fragmentation options. The important findings were that QM/MM optimization was preferable to obtain reliable hydrated structures of the ligands, that the 3-body correction was important for quantitative evaluation of the solvation energy, and that the dehydration effect was most remarkable near the hydrophobic sections of the ligands. In addition, the hydration energy calculated by the explicit solvent was compared with the hydration free energy calculated by the implicit solvent via the Poisson-Boltzmann equation, and the two showed a fairly good correlation. These findings will serve as useful information for rapid drug design.

Ab initio fragment molecular orbital (FMO) method applied to analysis of the ligand-protein interaction in a pheromone-binding protein.

Full quantum computation of the electronic state of proteins has recently become possible by the advent of the ab initio fragment molecular orbital (FMO) method. We applied this method to the analysis of the interaction between the Bombyx mori pheromone-binding protein and its ligand, bombykol. The protein-ligand interaction of this molecular complex was minutely analyzed by the FMO method, and the analysis revealed several important interactions between the ligand and amino acid residues.

Molecular dynamics simulations revealed Ca(2+)-dependent conformational change of Calmodulin.

Molecular dynamics simulations were performed to simulate Ca(2+)-dependent conformational change of calmodulin (CaM). Simulations of the fully Ca(2+)-bound form of CaM (Holo-CaM) and the Ca(2+)-free form (Apo-CaM) were performed in solution for 4 ns starting from the X-ray crystal structure of Holo-CaM. A striking difference was observed between the trajectories of Holo-CaM and Apo-CaM: the central helix remained straight in the former but became largely bent in the latter. Also, the flexibility of Apo-CaM was higher than that of Holo-CaM. The results indicated that the bound Ca(2+) ions harden the structure of CaM.

PEACH 4 with ABINIT-MP: a general platform for classical and quantum simulations of biological molecules.

A program package for molecular simulations of biological molecules was developed. The package, "PEACH version 4 with ABINIT-MP version 20021029," was constructed by incorporating ABINIT-MP, a program for the fragment molecular orbital (FMO) method [Chem.Phys. Lett. 313 (1999) 701], into PEACH, a program package for classical molecular dynamics simulations (MD). A few capabilities of the package were demonstrated. First, high parallel efficiency of FMO was demonstrated in a single point calculation of a protein. Second,FMO-MD simulations [Chem. Phys. Lett. 372 (2003) 342] of a peptide were performed with and without explicit solvent, and the simulations showed the influence of the solvent on the electronic state of the peptide.

Change in a protein's electronic structure induced by an explicit solvent: an ab initio fragment molecular orbital study of ubiquitin.

The effect of solvation on the electronic structure of the ubiquitin protein was analyzed using the ab initio fragment molecular orbital (FMO) method. FMO calculations were performed for the protein in vacuo, and the protein was immersed in an explicit solvent shell as thick as 12 A at the HF or MP2 level by using the 6-31G* basis set. The protein's physical properties examined were the net charge, the dipole moment, the internal energy, and the solvent interaction energy. Comparison of the computational results revealed the following changes in the protein upon solvation. First, the positively charged amino acid residues on the protein surface drew electrons from the solvent, while the negatively charged ones transfer electrons to the solvent. Second, the dipole moment of the protein was enhanced as a result of the polarization. Third, the internal energy of the protein was destabilized, but the destabilization was more than compensated for by the generation of a favorable protein-solvent interaction. Finally, the energetic changes were elicited both by the electron correlation effect of the first solvent shell and by the electrostatic effect of more distant solvent molecules. These findings were consistent with the picture of the solvated protein being a polarizable molecule dissolved in a dielectric media.