Adoption of CRISPR–Cas systems, such as CRISPR–Cas9 and CRISPR–Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense (‘Cascade’: Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR–Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR–Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR–Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

We designed and synthesized a medium-firm drug-candidate library of cryptand-like structures possessing a randomized peptide linker on the bacteriophage T7. From the macrocyclic library with a 109 diversity, we obtained a binder toward a cancer-related protein (Hsp90) with an antibody-like strong affinity (KD = 62 nM) and the binding was driven by the enthalpy. The selected supramolecular ligand inhibited Hsp90 activity by site-specific binding outside of the well-known ATP-binding pocket on the N-terminal domain (NTD).

Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the β2-microglobulin (β2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized β2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with β2m, thus accounting for β2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.

Robust and reliable analyses of long trajectories from molecular dynamics simulations are important for investigations of functions and mechanisms of proteins. Structural fitting is necessary for various analyses of protein dynamics, thus removing time-dependent translational and rotational movements. However, the fitting is often difficult for highly flexible molecules. Thus, to address the issues, we proposed a fitting algorithm that uses the Bayesian inference method in combination with rotational fitting-weight improvements, and the well-studied globular protein systems trpcage and lysozyme were used for investigations. The present method clearly identified rigid core regions that fluctuate less than other regions and also separated core regions from highly fluctuating regions with greater accuracy than conventional methods. Our method also provided simultaneous variance-covariance matrix elements composed of atomic coordinates, allowing us to perform principle component analysis and prepare domain cross-correlation map during molecular dynamics simulations in an on-the-fly manner. Published by AIP Publishing.

This paper reviews various enhanced conformational sampling methods and explicit/implicit solvent/membrane models, as well as their recent applications to the exploration of the structure and dynamics of membranes and membrane proteins. Molecular dynamics simulations have become an essential tool to investigate biological problems, and their success relies on proper molecular models together with efficient conformational sampling methods. The implicit representation of solvent/membrane environments is reasonable approximation to the explicit all-atom models, considering the balance between computational cost and simulation accuracy. Implicit models can be easily combined with replica-exchange molecular dynamics methods to explore a wider conformational space of a protein. Other molecular models and enhanced conformational sampling methods are also briefly discussed. As application examples, we introduce recent simulation studies of glycophorin A, phospholamban, amyloid precursor protein, and mixed lipid bilayers and discuss the accuracy and efficiency of each simulation model and method. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. (C) 2016 The Authors. Published by Elsevier B.V.

Replica-exchange molecular dynamics (REMD) method is one of the enhanced conformational sampling algorithms widely applied in computational biophysics and biochemistry. In the method, molecular dynamics (MD) simulations for multiple replicas of a target system are performed simultaneously and independently. Every few steps, temperatures or other parameters are exchanged between a pair of replicas according to the modified Metropolis criteria. Replica-Exchange INterface (REIN) is interface software for REMD simulations. It utilizes existing MD software without modification and performs the exchanges of replicas. In this article, we introduce the software design of REIN and demonstrate its applicability through benchmark simulations of alanine pentapeptide in explicit water, as well as the free-energy analysis of N-glycan and Tom20-presequence complex in solution. (c) 2014 Wiley Periodicals, Inc.

Structural information of a transmembrane (TM) helix dimer is useful in understanding molecular mechanisms of important biological phenomena such as signal transduction across the cell membrane. Here, we describe an umbrella sampling (US) scheme for predicting the structure of a TM helix dimer in implicit membrane using the interhelical crossing angle and the TM-TM relative rotation angles as the reaction coordinates. This scheme conducts an efficient conformational search on TM-TM contact interfaces, and its robustness is tested by predicting the structures of glycophorin A (GpA) and receptor tyrosine kinase EphA1 (EphA1) TM dimers. The nuclear magnetic resonance (NMR) structures of both proteins correspond to the global free-energy minimum states in their free-energy landscapes. In addition, using the landscape of GpA as a reference, we also examine the protocols of temperature replica-exchange molecular dynamics (REMD) simulations for structure prediction of TM helix dimers in implicit membrane. A wide temperature range in REMD simulations, for example, 250-1000 K, is required to efficiently obtain a free-energy landscape consistent with the US simulations. The interhelical crossing angle and the TM-TM relative rotation angles can be used as reaction coordinates in multidimensional US and be good measures for conformational sampling of REMD simulations. (C) 2013 Wiley Periodicals, Inc.

Tom20 is located at the outer membrane of mitochondria and functions as a receptor for the N-terminal presequence of mitochondrial-precursor proteins. Recently, three atomic structures of the Tom20-presequence complex were determined using X-ray crystallography and classified into A-, M-, and Y-poses in terms of their presequence-binding modes. Combined with biochemical and NMR data, a dynamic-equilibrium model between the three poses has been proposed. To investigate this mechanism in further detail, we performed all-atom molecular dynamics (MD) simulations and replica-exchange MD (REMD) simulations of the Tom20-presequence complex in explicit water. In the REMD simulations, one major distribution and another minor one were observed in the converged free-energy landscape at 300 K. In the major distribution, structures similar to A- and M-poses exist, whereas those similar to Y-pose are located in the minor one, suggesting that A-pose in solution is more stable than Y-pose. A k-means clustering algorithm revealed a new pose not yet obtained by X-ray crystallography. This structure has double salt bridges between Arg14′ in the presequence and Glu78 or Glu79 in Tom20 and can explain the binding affinity of the complex in previous pull-down assay experiments. Structural clustering and analyses of contacts between Tom20 and the presequence suggest smooth conformational changes from Y- to A-poses through low activation barriers. M-pose lies between Y- and A-poses as a metastable state. The REMD simulations thus provide insights into the energetics of the multiple-binding forms and help to detail the progressive conformational states in the dynamic-equilibrium model based on the experimental data. © 2013 American Chemical Society.

The introduction of bisecting GlcNAc and core fucosylation in N-glycans is essential for fine functional regulation of glycoproteins. In this paper, the effect of these modifications on the conformational properties Of N-glycans is examined at the atomic level by performing replica exchange molecular dynamics (REMD) simulations. We simulate four biantennary complex type N-glycans, namely, unmodified, two single substituted with either bisecting GlcNAc or core fucose, and disubstituted forms. By using REMD as an enhanced sampling technique, five distinct conformers in solution, each of which is characterized by its local orientation of the Man alpha 1-6Man glycosidic linkage, are observed for all four N-glycans. The chemical modifications significantly change their conformational equilibria. The number of major conformers is reduced from five to two and from five to four upon the introduction of bisecting GlcNAc and core fucosylation; respectively, The population change is attributed to specific inter residue hydrogen bonds, including water mediated Ones. The experimental NMR data, including nuclear Overhauser enhancement and scalar J-coupling constant's, are well reproduced; taking the multiple conformers into account Our structural model supports the concept of "conformer selection"; which emphasize the, conformational flexibility of N-glycans in protein-glycan interactions.

Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor virus, encodes two homologous membrane-associated E3 ubiquitin ligases, modulator of immune recognition 1 (MIR1) and MIR2, to evade host immunity. Both MIR1 and MIR2 downregulate the surface expression of major histocompatibility complex class I (MHC I) molecules through ubiquitin-mediated endocytosis followed by lysosomal degradation. Since MIR2 additionally downregulates a costimulatory molecule (B7-2) and an integrin ligand (intercellular adhesion molecule 1 [ICAM-1]), MIR2 is thought to be a more important molecule for immune evasion than MIR1; however, the molecular basis of the MIR2 substrate specificity remains unclear. To address this issue, we determined which regions of B7-2 and MIR2 are required for MIR2-mediated B7-2 downregulation. Experiments with chimeras made by swapping domains between human B7-2 and CD8 alpha, a non-MIR2 substrate, and between MIR1 and MIR2 demonstrated a significant contribution of the juxtamembrane (JM) region of B7-2 and the intertransmembrane (ITM) region of MIR2 to MIR2-mediated downregulation. Structure prediction and mutagenesis analyses indicate that Phe119 and Ser120 in the MIR2 ITM region and Asp244 in the B7-2 JM region contribute to the recognition of B7-2 by MIR2. This finding provides new insight into the molecular basis of substrate recognition by MIR family members.

Protein-glycan recognition regulates a wide range of biological and pathogenic processes. Conformational diversity of glycans in solution is apparently incompatible with specific binding to their receptor proteins. One possibility is that among the different conformational states of a glycan, only one conformer is utilized for specific binding to a protein. However, the labile nature of glycans makes characterizing their conformational states a challenging issue. All-atom molecular dynamics (MD) simulations provide the atomic details of glycan structures in solution, but fairly extensive sampling is required for simulating the transitions between rotameric states. This difficulty limits application of conventional MD simulations to small fragments like di- and tri-saccharides. Replica-exchange molecular dynamics (REMD) simulation, with extensive sampling of structures in solution, provides a valuable way to identify a family of glycan conformers. This article reviews recent REMD simulations of glycans carried out by us or other research groups and provides new insights into the conformational equilibria of N-glycans and their alteration by chemical modification. We also emphasize the importance of statistical averaging over the multiple conformers of glycans for comparing simulation results with experimental observables. The results support the concept of "conformer selection" in protein-glycan recognition. © 2012 International Union for Pure and Applied Biophysics (IUPAB) and Springer.

Structural diversity of N-glycans is essential for specific binding to their receptor proteins. To gain insights into structural and dynamic aspects in atomic detail not normally accessible by experiment, we here perform extensive molecular-dynamics simulations of N-glycans in solution using the replica-exchange method. The simulations show that five distinct conformers exist in solution for the N-glycans with and without bisecting GlcNAc. Importantly, the population sizes of three of the conformers are drastically reduced upon the introduction of bisecting GlcNAc. This is caused by a local hydrogen-bond rearrangement proximal to the bisecting GlcNAc. These simulations show that an N-glycan modification like the bisecting GlcNAc selects a certain "key" (or group of "keys") within the framework of the "bunch of keys" mechanism. Hence, the range of specific glycan-protein interactions and affinity changes need to be understood in terms of the structural diversity of glycans and the alteration of conformational equilibria by core modification.

AcrB is a membrane protein and a multidrug efflux transporter. Although the recently solved X-ray crystal structures of AcrB provide a rough sketch of how drugs efflux, the pathway and mechanism have not been completely elucidated. In this study, a ligand-mapping method based on the 3D-RISM molecular theory of solvation, which we recently developed, is applied to AcrB in order to identify the drug efflux pathway. We use a fragment-based approach as a strategy to map chemical functionality on the internal surfaces. A few "multifunctional" ligand-binding sites, which recognize various types of functional groups, are detected inside the porter domain. Spatial links between the multifunctional sites indicate a probable multidrug efflux pathway. The frustrated environment of the protein cavity constructed of weak interactions between ligand and protein may be a mechanism for allowing smooth transportation through the protein. Guided diffusion appears to be the main mechanism for efflux.

The generation of T cell receptor (TCR) sequence diversity can produce forbidden clones able to recognize self-antigens Here the structure of the complex between a myelin basic protein peptide (MBP85-99) human leukocyte antigen (HLA)-DR2 (DRB1*1501/DRA) and TCR-Ob 2F3 the dominant autoimmune clone obtained from a multiple sclerosis (MS) patient has been determined using structural docking simulation and dynamics in silico and compared to the structure of TCR-Ob 1A12 complexes with the same MHC/peptide determined by X-ray crystallography The two TCRs differ by three amino acids in the CDR3 alpha and beta loops As the result different hydrogen bonds are formed between the two CDR3 beta loops and the peptide in the complexes of the simulated structures with three hydrogen bonds seen in the TCR-Ob 2F3 complex and five in the TCR-Ob 1A12 complex The two TCRs each located near the N-terminal end of the HLA-DR2 binding groove and both had an orthogonal binding axis but they deviated by about 10 Simulation methods such as structural docking and molecular dynamics as used here provide an avenue to understand molecular binding mode efficiently and more rapidly than obtaining multiple crystal structures when a large structural database is already available (C) 2010 Elsevier Ltd All rights reserved

我々は方法論やプログラムを開発しながら,疾患や生体機構に関連する生体分子の機能解明の為の研究を中規模-大規模並列型計算機を用いて行っている.現在,アルツハイマー病の機構解明に関する研究を行なっており,アミロイド β ペプチドの生成機構を関連蛋白質の構造予測から解明しようとしている.具体的にはレプリカ交換分子動力学法 (REMD) という効率の良いサンプリング手法を使い,生体膜中のアミロイド前駆体蛋白質 (APP) の構造予測を行っている.この計算により,アルツハイマー病の初期過程がわかってきたので報告する.

Aggregation of amyloid beta peptide (An) in the brain is the primary element in the pathogenesis of Alzheimer's disease. A beta is derived from amyloid precursor protein (APP) in the membrane due to the cleavages by beta- and gamma-secretases. Here, we predict the transmembrane structures of the wild-type and mutant APP in the biological membrane by replica-exchange molecular dynamics simulations. The simulations illustrate large conformational differences between the wild type and mutant APP fragments in the membrane. Dimerization of the wild type occurs due to the C alpha-H center dot center dot center dot O hydrogen bonds at the Gly-XXX-Gly motifs between two APP fragments, whereas the mutant dimer is stabilized by the interactions between hydrophobic side chains. We also observe the downward shift of gamma-cleavage site in the mutant APP, which may cause the prohibition of A beta production.

Aggregation of Amyloid beta (A beta) peptide has been linked to the neurodegenerative Alzheimer's Disease and implicated in other amyloid diseases including cerebral amyloid angiopathy. A beta peptide is generated by cleavage of the amyloid precursor protein (APP) by transmembrane proteases. It is crucial to determine the structures of beta-amyloid peptides in a membrane to provide a molecular basis for the cleavage mechanism. We report the structures of amyloid beta peptide (A beta(1-40) and A beta(1-42)) as well as the 672-726 fragment of APP (referred to as A beta(1-55)) in a membrane environment determined by replica-exchange molecular dynamics simulation. A beta(1-40) is found to have two helical domains A (13-22) and B(30-35) and a type I beta-turn at 23-27. The peptide is localized at the interface between membrane and: solvent. Substantial fluctuations in domain A are observed. The dominant simulated tertiary structure of A beta(1-40) is observed to be similar to the simulated A beta(1-42) structure. However, there are differences observed in the overall conformational ensemble, as characterized by the two-dimensional free energy surfaces. The fragment of APP (A beta(1-55)) is observed to have a long transmembrane helix. The position of the transmembrane region and ensemble of membrane structures are elucidated. The conformational transition between the transmembrane A beta(1-55) structure, prior to cleavage, and the A beta(1-40) structure, following cleavage, is proposed.

AB peptide is an essential protein in the pathogenesis of Alzheimer's disease and is derived from amyloid precursor protein (APP) in the membrane by beta- and gamma-secretase cleavage. An experimental study has shown that a pairwise replacement of Gly with Leu in APP enhances homodimerization but Leads to a drastic reduction of AB secretion. To resolve this apparent discrepancy, we predicted the wild-type (WT) and mutant APP dimer conformations by replica-exchange molecular dynamics simulations using the implicit membrane model IMM1. The simulations illustrate large conformational differences between the WT and mutant APP fragments in the membrane. Dimerization of the WT is due to two C(alpha)-H center dot center dot center dot O hydrogen bonds between two APP fragments, whereas dimerization of the mutant is due to hydrophobic interactions. In the mutant, each APP fragment is more tilted, and the gamma-cleavage site is shifted toward the center of the membrane. This position produces a mismatch between the active site of gamma-secretase and the gamma-cteavage site of APP that might prohibit AB production.

Phospholamban is a 52-residue integral membrane protein that regulates the activity of the sarcoplasmic reticulum calcium pump in cardiac muscle. Its inhibitory action is relieved when phospholamban is phosphorylated at Ser16 by cAMP-dependent protein kinase. To computationally explore all possible conformations of the phosphorylated form, and thereby to understand the structural effects of phosphorylation, replica-exchange molecular dynamics (REMD) was applied to the cytoplasmic domain that includes Ser16. The simulations showed that (i) without phosphorylation, the region from Lys3 to Ser16 takes all alpha-helical conformations; (ii) when phosphorylated, the alpha-helix is partially unwound in the C-terminal part ( from Ser10 to Ala15) resulting in less extended conformations; (iii) the phosphate at Ser16 forms salt bridges with Arg9, Arg13, and/or Arg14; and (iv) the salt bridges with Arg13 and Arg14 distort the alpha-helix and induce unwinding of the C-terminal part. We then applied conventional all-atom molecular dynamics simulations to the full-length phospholamban in the phospholipid bilayer. The results were consistent with those obtained with REMD simulations, suggesting that the transmembrane part of phospholamban and the lipid bilayer itself have only minor effects on the conformational changes in the cytoplasmic domain. The distortions caused by the salt bridges involving the phosphate at Ser16 readily explain the relief of the inhibitory effect of phospholamban by phosphorylation, as they will substantially reduce the population of all helical conformations, which are presumably required for the binding to the calcium pump. This will also be the mechanism for releasing the phosphorylated phospholamban from kinase.

Ca2+-ATPase of sarcoplasmic reticulum is an ATR-powered Ca2+ pump but also a H+ pump in the opposite direction with no demonstrated functional role. Here, we report a 2.4-angstrom-resolution crystal structure of the Ca2+ -ATPase in the absence of Ca2+ stabilized by two inhibitors, dibutyidihydroxybenzene, which bridges two transmembrane helices, and thapsigargin, also bound in the membrane region. Now visualized are water and several phospholipid molecules, one of which occupies a cleft between two transmembrane helices. Atomic models of the Ca2+ binding sites with explicit hydrogens derived by continuum electrostatic calculations show how water and protons fill the space and compensate charge imbalance created by Ca2+-release. They suggest that H+ countertransport is a consequence of a requirement for maintaining structural integrity of the empty Ca2+-binding sites. For this reason, cation countertransport is probably mandatory for all P-type ATPases and possibly accompanies transport of water as well.

Real-time dynamics of charge density and lattice displacements is studied during photoinduced ionic-to-neutral phase transitions by using a one-dimensional extended Peierls-Hubbard model with alternating potentials for the one-dimensional mixed-stack charge-transfer complex, TTF-CA. The time-dependent Schrodinger equation and the classical equation of motion are solved for the electronic and lattice parts, respectively. We show how neutral domains grow in the ionic background. As the photoexcitation becomes intense, more neutral domains are created. Above threshold intensity, the neutral phase is finally achieved. After the photoexcitation, ionic domains with wrong polarization also appear. They quickly reduce the averaged staggered lattice displacement, compared with the averaged ionicity. As the degree of initial lattice disorder increases, more solitons appear between these ionic domains with different polarizations, which obstruct the growth of neutral domains and slow down the transition.

A photoinduced transition from a charge-density-wave (CDW) phase to a charge-polarization (CP) phase has been recently found in a one-dimensional halogen-bridged binuclear platinum complex R-4[Pt-2(pop)(4)I]nH(2)O [pop=P2O5H22-, R=(C2H5)(2)NH2]. Its mechanism is theoretically studied by solving the time-dependent Hartree-Fock equation for a one-dimensional two-band three-quarter-filled Peierls-Hubbard model. Above a threshold in the photoexcitation intensity, a transition takes place from the CDW to CP phases. The threshold intensity depends on the relative stability of these phases, which can be explained qualitatively by their diabatic potentials. However, the transition from the CP to CDW phases is hardly realized for two reasons: (i) low-energy charge-transfer processes occur only within a binuclear unit in the CP phase; (ii) it is difficult for the CDW order to become long ranged owing to its weak coherence. The effective transfer integrals required for the coherence are evaluated.

Dynamics of the ionicity after photoexcitations in mixed-stack charge-transfer complexes is numerically studied by using a one-dimensional extended Peierls-Hubbard model with alternating potentials at half filling. The time-dependent Schrodinger equation and the Newton equation are solved for the electronic and lattice parts, respectively. Fourier analysis is performed for the ionicity. During the photoinduced phase transition, slow components are dominant and very broad reflecting complex motion of domain walls and lattice displacements. After the transition, fast and slow oscillations are clearly seen, which correspond to the electronic and lattice motion, respectively.

In the one-dimensional two-band three-quarter-filled Peierls-Hubbard model for halogen-bridged binuclear metal complexes R-4[Pt-2(pop)(4)I]nH(2)O with counter ion R and pop=P2O5H22-, a transition from the charge-density-wave phase to the charge-polarization phase is photoinduced, but the opposite process is not. Its origin is explained by considering differences in low-energy photoexcitations in the two phases and coherence of respective order parameters. The different dynamics is demonstrated by solving the time-dependent Schrodinger equation.

Real-time dynamics of domain walls between the neutral and ionic phases just after photoexcitations is studied by fully solving the time-dependent Schrodinger equation for a one-dimensional extended Peierls-Hubbard (PH) model, not by relying on the adiabatic approximation. The unrestricted Hartree-Fock (HF) approximation is used for electrons, and the lattice displacements are treated classically. Three characteristic time scales are observed: rapid oscillation of ionicity owing to the local charge transfer; slow oscillation of lattice displacements; and even slower and collective motion of domain walls. Steady growth of a metastable domain is achieved after complicated competition of micro domains. The relevance to recently measured, time-resolved photoreflectance spectra in TTF-CA is discussed.

We study i) the ground-state and optical-excitation properties of halogen-bridged binuclear metal complexes, which are known as MMX chain compounds, by the strong-coupling expansion for a one-dimensional two-orbital extended Peierls-Hubbard model, and ii) the dynamics of domain walls between the neutral and ionic phases in mixed-stack charge-transfer complexes, by solving the time-dependent Schrodinger equation for a one-dimensional extended Peierls-Hubbard model with alternating energy levels. We find in i) that competition between electron-electron and electron-lattice interactions and competition between short- and long-range interactions are both important for the electronic phase variation, and in ii) that charge and lattice dynamics immediately after photoexcitations is complex and not explained simply within the domino picture.

Electron-impact ionization of the collinear [Formula Presented] model atom is investigated in order to examine a nonclassical behavior of the ionization cross sections. Slightly above the ionization threshold, ab initio calculations reveal a distinctive dip due to short-range dynamics. The dip is a strongly energy-dependent feature in the usually smooth and structureless ionization cross section and is foreign to treatments based on classical, as well as semiclassical mechanics. The [Formula Presented] model thus serves as a counterexample to the standard Wannier treatment of near-threshold ionization. The hyperspherical hidden-crossing theory is applied to identify the origin of the dip. © 1999 The American Physical Society.

Relying on an entirely ab initio quantum-mechanical scheme, we investigate the threshold behavior of a model electron-impact ionization problem in which the target hydrogen atom interacts with the incident electron only by monopole. The total ionization cross section for a singlet is shown to follow the threshold law of an exponential form as proposed by Macek and Ihra [Phys. Rev. A 55, 2024 (1997)], thus supporting the argument based on the local instability of the “ridge” motion despite the reported absence of classical ridge trajectories. Below the classical threshold, quantum diffraction allows the two electrons to have a large probability amplitude in the region inaccessible to the classical trajectories. The energy distribution for singlet in final continuum channels is shown to have a hitherto unexpected V-shaped structure at energies between 0 and 1 a.u. above the ionization threshold. The V structure becomes sharper toward the threshold while it approaches the quadratic form surmised by Bray [Phys. Rev. Lett. 78, 4721 (1997)] at higher energies. © 1999 The American Physical Society.

近年、計算科学に関する技術の進歩によりコンピュータシステムの性能が劇的に上がってきた。この様なスーパーコンピュータをはじめとするコンピュータシステムを利用する事で、精度の高い様々な生体分子シミュレーションが可能になってきた。本講演では前半に最近のスーパーコンピュータの話をし、後半に講演者が行っている生体分子シミュレーションを用いたアルツハイマー病の分子機構に関する研究の紹介をする。

ウエストナイルウイルス（WNV）は、時に致死性の脳炎・髄膜炎を惹き起こすフラビウイルス科の蚊媒介性ヒト感染性ウイルスである。世界の広い地域に分布し我が国への感染拡大の危険性が指摘されているが、実用的な治療薬やワクチンは未だ無い。研究分担者らによって見出された WNV に働く中和抗体は、同じフラビウイルス科の日本脳炎ウイルス(JEV)とも交差反応を示すという興味深い性質を持つ。我々はその抗体と WNV のエンベロープタンパク質（Ｅタンパク質）との複合体の立体構造の決定に成功し、異なるウイルスを同時に認識する抗体の分子機構を明らかにした。その構造基盤を発展させ、(1) フラビウイルス科の各種ウイルスを同時に認識する抗体を開発し、異なるフラビウイルス感染症のどれにも効果のある抗体医薬開発につながる知見を得ること、(2) フラビウイルス科の中の特定のウイルスだけを認識する抗体を開発し、多重感染地域などでどのウイルスに感染しているかを判定できる診断薬の開発につながる知見を得ること、を研究の目的とし、フラビウイルス科の日本脳炎、黄熱病、ジカ熱の各ウイルス（JEV, YFV, ZIKV）のＥタンパク質とその中和抗体の認識機構を明らかにすべく、両者のタンパク質複合体の立体構造解析を目指し研究を行った。

TBAは天然の４種の核酸の中のTとGのみで形成され、塩基配列は5'-GGTTGGTGTGGTTGG-3'である。４つのグアニン対を持ち、２つの対の各々が安定なグアニン４重鎖と呼ばれる平面構造（G-plate、G-quadruplex）を形成する。２つの平面が並行なチューブ状に並び、その中にK+イオンを捕獲するイオンチャネルを形成すると堅固なDNAアプタマーとなる。G-plateによる堅固な構造は生体内では染色体の末端部にあるテロメアにも見られ、癌の抑制/惹起の研究でも興味を持たれている。本研究はこの興味深いDNAアプタマーの(a)構造と安定性、(b)ヌクレアーゼ耐性、(c)特異的結合能点を考察する。また、共同研究者らから動力学の分析に有効な数理手法やTBAに関連するDNAアプタマーの実験的研究の情報を得ながら研究を進めている。 全体像を得るために特に重要なものは(a)である。TBAだけではなく、類似の構造で性質の異なるものの比較が必要である。実験的先行研究では、幾つかの核酸をα-アノマーで置き換えているが、アノマーの実験的・理論的構造決定はできていない。MD計算をするため、山越と渡辺はxleapを利用し、宮下はpythonツールを開発して、アノマー化したTBAのpdbを作った。塩基を糖との軸について回転させてsynとantiの配置による安定性の吟味もMDシミュレーションで実施したが、これらの試行的な結果はまだ発表の段階に至っていない。 本研究の目的(b)に関して、瀧はTBAにターゲットと共有結合するwarheadを付けたDNA・コバレント・アプタマー(TBDcA)を実験的に作成し、その各種ヌクレアーゼ耐性機構を検討し、有意な結果を得た。宮下はTBDcAの理論計算の前段階としてペプチドをベースにしたペプチド・コバレント・アプタマー（PecA）の知見を得る準備研究を実施した。

アルツハイマー病の初期過程における分子機構では、ニューロン細胞にある膜タンパク質であるアミロイド前駆体タンパク質（APP）が通常では膜タンパク質のα切断酵素によって切断されるところが、膜タンパク質のβ切断酵素によって切断されC-APPが生成される。その後、γ切断酵素によって切断され、アミロイドβペプチドが産生される。本研究では、分子シミュレーションを用いたアルツハイマー病におけるAβ産生の起点機構の詳細解明における、特にβ切断酵素とAPPとの特異的な動的相互作用の観点から研究を進めている。 我々のこれまでの研究から、生体分子の相互作用には特異的な動的相互作用があることに気づいた。これは例えば膜タンパク質間の相互作用の際にはコレステロールなどの脂質分子が相互作用に関わってきたりする。これまでの研究では膜貫通部位のみの動力学を考慮していたが、本研究では全長を考える点が新しい点である。本研究はβ切断酵素とAPP、α切断酵素とAPPとの相互作用の研究であるが、このようなアルツハイマー病の起点現象を通して、膜タンパク質間相互作用の知見を深めることにつなげようとしている。 本年度成果としては、１）β切断酵素の全長構造のモデリングを実施し、２）その全原子モデル分子動力学（MD）シミュレーションを実施した。その結果、細胞外領域にあるβ切断酵素の切断部位の運動には制限があり、β切断酵素とAPPとの結合過程が切断に影響することがわかってきた。

大きな膜タンパク質と小さな膜タンパク質のドッキングインターフェースを見つけるためのプログラムモジュールの開発を実施した。直接の目的としては膜タンパク質であるγ切断酵素とアミロイド前駆体タンパク質(APP)のドッキングインターフェースを見つけるために作成した。 ①当初の次善策であるNAMDを用いたモジュールを作成した。現在、その公開も考えている。②ターゲットとなるγ切断酵素のシミュレーションを実施し、変異によりそのタンパク質の運動に大きな違いが生じる事がわかった。③作成したプログラムモジュールをγ切断酵素とAPPのドッキングシミュレーションに使用した。現在、詳細な解析を実施中である。

生体膜は様々な脂質分子が混ざってできている。またラフトと呼ばれる、コレスレロール脂質を多く含むマイクロドメインを生体膜中に作る事が知られており、これが膜タンパク質の機能に強く影響を与えている事がわかってきた。本研究課題の目標は（１）混合膜中のタンパク質に関する研究及び（２）複合（混合）膜中のタンパク質のモデリングプロトコル開発である。平成２４年度は、主に、混合膜、粗視化モデルから段階的に解像度を上げて全原子モデルにするプロトコルを作成し、関連プログラムの作成を実施した。分子間相互作用の詳細を調べる為には、全原子モデルと呼ばれる原子1個が粒子1個に対応する解像度でのシミュレーションを実施する必要がある。しかし、全原子モデル分子動力学（MD)シミュレーションで、たとえ非常に多くの計算資源を使ったとしても複数の脂質分子を混ぜる事は非常に難しい。それは通常のコンピュータクラスタでの全原子モデルMDシミュレーションが脂質分子の拡散の時間スケールにまだ追いついていない為である。またランダムに脂質を配置する方法もあるが、得られた配置がふさわしいか疑問である。そこで粗視化モデルを用いて解決する方法の提案を行った。粗視化モデルからunited atomモデル、全原子モデルへと段階的に変換するプロトコルを作成した。現在、混合膜中のアルツハイマー病の原因タンパク質と脂質分子との相互作用を調べる為のシミュレーションを実施中である。これまで単一膜中の膜タンパク質のシミュレーション研究が多かったが、今後必要性が増してくるであろう混合膜中での膜タンパク質の研究の為に、このプロトコルは非常に有用となるだろう。今後（２５年度中に）、混合膜中の膜タンパク質のシミュレーション研究の論文でこのプロトコル内容も発表する予定である。

　テレビ・ラジオ番組

　テレビ・ラジオ番組