Publications2007-2011

<Background>
Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly.
<Results>
To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups.
<Conclusions>
We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein’s specific molecular function.

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.

Reversible phosphorylation of proteins is a post-translational modification that regulates diverse biological processes. The molecular mechanism underlying phosphoryl transfer catalyzed by enzymes, in particular the nature of transition state (TS), remains a subject of active debate. Structural evidence supports an associative TS, whereas physical organic studies point to a dissociative character. In this article, we briefly introduce our recent effort using the hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to resolve the controversy. We perform QM/MM simulations for the reversible phosphorylation of phosphoserine phosphatase (PSP), which belongs to one of the largest phosphotransferase families characterized to data. Both phosphorylation and dephosphorylation reactions are investigated based on the two-dimensional energy surfaces along phosphoryl and proton transfer coordinates. The resultant structures of the active site at TS in both reactions have compact geometries but a less electron density of the phosphoryl group. This suggests that the TS of PSP has a geometrically associative yet electronically dissociative character and strongly depends on proton transfer being coupled with phosphoryl transfer. Structure and literature database searches on phosphotransferases suggest that such a hybrid TS is consistent with many structures and physical organic studies and likely holds for most enzymes catalyzing phosphoryl transfer.

A series of magnesium fluorides MgF_n^2-n, multiply charged anions, in the gas phase and in aqueous solution were theoretically studied with a hybrid approach of quantum chemistry and statistical mechanics, called RISM-SCF-SEDD theory. In the gas phase, MgF₃^– is the most stable species among the complexes (n = 1 – 6). In contrast, due to compensation between the intramolecular energy and solvation free energy, the stabilities of a number of complexes with different n are comparable in aqueous solution. Based on accurate evaluation of free energy change, the mole fraction of MgF₄^2- is the highest in the range from pF = 2.0 to 3.0 of aqueous solution. This is consistent with the available PDB data of the enzymes that catalyze the phosphoryl transfer reactions. The hydration structures of magnesium fluorides obtained by RISM-SCF-SEDD theory provide insight into their structural changes from the gas phase to aqueous solution.

AcrB is a membrane protein acting as a multidrug efflux transporter. Although the recently-solved X-ray crystal structures of AcrB provided a rough sketch for the drug efflux mechanism, the pathway has not been completely elucidated in atomic resolution. In this study, a ligand-mapping method based on the molecular theory of solvation, which has been recently developed by ourselves, is applied to AcrB in order to identify the drug efflux pathway. As an effective strategy, a fragment-based approach is adopted to map chemical functionality on the internal surfaces. As a result, a few “multifunctional” ligand-binding sites, which recognize various types of functional groups, are detected inside the porter domain. A spatial link between the multi-functional sites indicates a probable multidrug efflux pathway. The chemical and physical driving forces to ingest and transport drugs are also discussed.

We investigate our free-energy function (FEF) for discriminating the native fold of a protein from misfolded decoys. It is a physics-based function using an all-atom model which comprises the hydration entropy (HE) and the total dehydration penalty (TDP). The HE is calculated using a hybrid of a statistical-mechanical theory applied to a molecular model for water and the morphometric approach. The energetic component is suitably taken into account in a simple manner as the TDP. On the basis of the results from a careful test of the FEF, which have newly been performed for 118 proteins in some representative decoy sets, we show that its performance is distinctly superior to that of any other function. By our FEF which precisely captures the features of the native structure, some important findings are made possible. For instance, our FEF varies largely from model to model for the candidate models obtained from nuclear magnetic resonance experiments. We can select the best model that is optimized in terms of the sum of the two components, HE and TDP. A decoy set is not suited to the test of a free-energy or potential function in cases where a protein isolated from a protein complex is considered and the structure in the complex is employed as the model NS of the isolated protein without any change or where portions of the terminus sides of a protein are removed and the percentage of the secondary structures lost due to the removal is significantly high.

Reversible phosphorylation of proteins is a post-translational modification that regulates diverse biological processes. The molecular mechanism underlying phosphoryl transfer catalyzed by enzymes remains a subject of active debate. In particular, the nature of transition state (TS), whether it has an associative or dissociative character, has been one of the most controversial issues. Structural evidence supports an associative TS, whereas physical organic studies point to a dissociative character. Here we perform hybrid quantum mechanics/molecular mechanics simulations for the reversible phosphorylation of phosphoserine phosphatase (PSP) to study the nature of the TS. Both phosphorylation and dephosphorylation reactions are investigated based on the two-dimensional energy surfaces along phosphoryl and proton transfer coordinates. The structures of the active site at TS in both reactions reveal compact geometries, consistent with crystal structures of PSP with phosphate analogues. On the other hand, the electron density of the phosphoryl group in both TS structures slightly decreases compared with that in the reactant states. These findings suggest that the TS of PSP has a geometrically associative yet electronically dissociative character and strongly depends on proton transfer being coupled with phosphoryl transfer. Structure and literature database searches on phosphotransferases suggest that such a hybrid TS is consistent with many structures and physical organic studies and likely holds for most enzymes catalyzing phosphoryl transfer.

The sarcoplasmic reticulum Ca²⁺-ATPase transports two Ca²⁺ per ATP hydrolyzed from the cytoplasm to the lumen against a large concentration gradient. During transport, the pump alters the affinity and accessibility for Ca²⁺ by rearrangements of transmembrane helices. In this study, all-atom molecular dynamics simulations were performed for wild type Ca²⁺-ATPase in the Ca²⁺-bound form and the Gln mutants of Glu771 and Glu908. Both of them contribute only one carboxyl oxygen to site I Ca²⁺, but only Glu771Gln completely looses the Ca²⁺-binding ability. The simulations show that: (i) For Glu771Gln, but not Glu908Gln, coordination of Ca²⁺ was critically disrupted. (ii) Coordination broke at site II first, although Glu771 and Glu908 only contribute to site I. (iii) A water molecule bound to site I Ca²⁺ and hydrogen bonded to Glu771 in wild type, drastically changed the coordination of Ca²⁺ in the mutant. (iv) Water molecules flooded the binding sites from the lumenal side. (v) The side chain conformation of Ile775, located at the head of a hydrophobic cluster near the lumenal surface, appears critical for keeping out bulk water. Thus the simulations highlight the importance of the water molecule bound to site I Ca²⁺ and point to a strong relationship between Ca²⁺-coordination and shielding of bulk water, providing insights into the mechanism of gating of ion pathways in cation pumps.

Many structural properties such as solvent accessibility, dihedral angles and helix-helix contacts can be assigned to each residue in a membrane protein. Independent studies exist on the analysis and sequence-based prediction of some of these so-called one-dimensional features. However, there is little explanation of why certain residues are predicted in a wrong structural class or with large errors in the absolute values of these features. On the other hand, membrane proteins undergo conformational changes to allow transport as well as ligand binding. These conformational changes often occur via residues that are inherently flexible and hence, predicting fluctuations in residue positions is of great significance.

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In spite of its small size, chicken villin headpiece subdomain HP36 folds into the native structure with a stable hydrophobic core within several microseconds. How such a small protein keeps up its conformational stability and fast folding in solution is an important issue for understanding molecular mechanisms of protein folding. In this study, we performed multicanonical replica-exchange simulations of HP36 in explicit water, starting from a fully extended conformation. We observed folding events of HP36 into the native-like conformations at least five times. The smallest backbone RMSD from the crystal structure was 1.1 Å. In the native-like conformations, the stably formed hydrophobic core was fully dehydrated. Statistical analyses of the simulation trajectories have shown the following sequential events in folding of HP36: (i) Helix 3 is formed at the earliest stage, (ii) the backbone and the side chains near the loop between Helices 2 and 3 take native-like conformations, and (iii) the side-chain packing at the hydrophobic core and the dehydration of the core side chains take place simultaneously at the later stage of folding. These suggest that the initial folding nucleus is not necessarily the same as the hydrophobic core. This is consistent with the recent experimental φ-value analysis.

An analytic gradient expression is formulated and implemented for the second-order Moller-Plesset perturbation theory (MP2) based on the generalized hybrid orbital QM/MM method. The method enables us to obtain an accurate geometry at a reasonable computational cost. The performance of the method is assessed for various isomers of alanine dipepetide. We also compare the optimized structures of fumaramide-derived [2]rotaxane and cAMP-dependent protein kinase with experiment.

Water penetration into the hydrophobic interior of proteins has been postulated to be a primary force driving pressure-induced denaturation of proteins. The water penetration model is supported by several theoretical and simulation studies, although its direct evidence is lacking. In this study, 1 microsecond all-atom molecular dynamics simulations of ubiquitin in explicit water at high and low pressures are performed to examine the water penetration model. The high-pressure simulation starts from a crystal structure at atmospheric pressure and successfully reproduces the main characteristics of a high-pressure structure obtained by NMR. Water penetrates into a specific hydrophobic core of the protein and is ejected from the interior several times. The structural transition results from the relative stabilization of a preexisting metastable structure by applying pressure. A time correlation analysis demonstrates that the transition is accompanied by the penetration of water within a time scale comparable to the relaxation time of water itself. Simultaneous water penetration only occurs above a certain high pressure.

The Sec translocon, a protein-conducting channel, consists of a heterotrimeric complex (SecYEG in bacteria and Sec61αβγ in eukaryotes) that provides a pathway for secretary proteins to cross membranes, or for membrane proteins to integrate into the membrane. The Sec translocon alone is a passive channel, and association with channel partners, including the ribosome or SecA ATPase in bacteria, is needed for protein translocation. Three recently published crystal structures of SecY are considered to represent the closed (resting state), pre-open (transitional state determined with the bound Fab fragment mimicking SecA interaction), and SecA-bound forms. To elucidate mechanisms of transition between closed and pre-open forms, we performed all-atom molecular dynamics simulations for the pre-open form of Thermus thermophilus SecYE and the closed form of Methanococcus janaschii SecYEβ in explicit solvent and membranes. We found that the closed form of SecY is stable, while the pre-open form without the Fab fragment undergoes large conformational changes toward the closed form. The pre-open form of SecY with Fab remains unchanged, suggesting that the cytosolic interaction mimicking SecA binding stabilizes the pre-open form of SecY. Importantly, a lipid molecule at the lateral gate region appears to be required to maintain the pre-open form in the membrane. We propose that the conformational transition from closed to pre-open states of SecY upon association with SecA facilitates intercalation of phospholipids at the lateral gate, inducing initial entry of the positively charged signal peptide into the channel.

The phosphoryl transfer reaction from phospho-L-serine (pSer), catalyzed by phosphoserine phosphatase, is investigated using the hybrid quantum mechanics/molecular mechanics calculations. The two-dimensional energy surface along the phosphoryl and proton transfer distances reveals early protonation of the leaving group oxygen of pSer, prior to the transition state (TS), which triggers subsequent phosphoryl transfer reaction. Calculated electronic properties of the phosphoryl group at the active site suggest significant metaphosphate-like character of TS, which is consistent with kinetic experiments on related phosphatases. The features are not obtained with a one-dimensional search along the phosphoryl transfer coordinate, due to inadequate description of proton movement.

We have developed a free-energy function based on an all-atom model for proteins. It comprises two components, the hydration entropy (HE) and the total dehydration penalty (TDP). Upon a transition to a more compact structure, the number of accessible configurations arising from the translational displacement of water molecules in the system increases, leading to a water-entropy gain. To fully account for this effect, the HE is calculated using a statistical-mechanical theory applied to a molecular model for water. The TDP corresponds to the sum of the hydration energy and the protein intramolecular energy when a fully extended structure, which possesses the maximum number of hydrogen bonds with water molecules and no intramolecular hydrogen bonds, is chosen as the standard one. When a donor and an acceptor (e.g., N and O, respectively) are buried in the interior after the break of hydrogen bonds with water molecules, if they form an intramolecular hydrogen bond, no penalty is imposed. When a donor or an acceptor is buried with no intramolecular hydrogen bond formed, an energetic penalty is imposed. We examine all the donors and acceptors for backbone-backbone, backbone-side chain, and side chain-side chain intramolecular hydrogen bonds and calculate the TDP. Our free-energy function has been tested for three different decoy sets. It is better than any other physics-based or knowledge-based potential function in terms of the accuracy in discriminating the native fold from misfolded decoys and the achievement of high Z-scores.

Aβ peptide is an essential protein in the pathogenesis of Alzheimer’s disease (AD) and is derived from Amyloid precursor protein (APP) in the membrane by β- and γ-secretase cleavage. An experimental study shows that a pairwise replacement of Gly with Leu in APP enhances homo dimerization, but leads to a drastic reduction of Aβ secretion. To resolve this apparent discrepancy, we predicted the wild type and the 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 the mutant APP fragments in the membrane. Dimerization of the wild type is due to the two C_α-H…O hydrogen bonds between two APP fragments, whereas dimerization of the mutant is due to the hydrophobic interactions. In the mutant, each APP fragment is more tilted and the γ-cleavage site was shifted toward the center of the membrane. This position produces a mismatch between the active site of γ-secretase and the γ-cleavage site of APP that might prohibit Aβ production.

Free-energy landscapes of proteins in solution are essential for understanding molecular mechanism of protein folding, stability, and dynamics. Because of the multiple-minima problem (or quasi-ergodicity problem), the conventional molecular dynamics or Monte Carlo methods cannot provide the landscapes accurately at low temperatures. By contrast, the simulations based on the generalized-ensemble algorithms can sample wider conformational spaces than the conventional approaches, thereby providing better free-energy landscapes of proteins at low temperatures. In this article, we review two well-known generalized-ensemble algorithms, namely, multicanonical algorithm and replica-exchange method, and then introduce further extensions of the above two methods, which are applicable to larger systems with rugged energy landscapes. These simulation methods have been applied to the protein folding simulations of the C-peptide in ribonuclease A with explicit solvent. We also demonstrate how the methods and the free-energy landscapes of proteins are useful for the biological research, by showing the simulation results on the phospholamban, a reversible regulator of sarco(end)plasmic reticulum Ca²⁺-pump.

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筋小胞体カルシウムポンプは、ATP加水分解によるエネルギーを利用して約10,000倍もの濃度勾配に逆らって、細胞質中に放出されたCa²⁺を筋小胞体内腔へと輸送する膜タンパク質である。近年、九つの反応中間体のX線結晶構造が明らかになり、カルシウムポンプの大規模な構造変化の能動輸送に果たす役割が明らかになった。我々は、この立体構造を用いた全原子分子動力学計算を行うことにより、Ca²⁺の輸送と対抗するプロトン輸送の機能的意義を明らかにした。また、カルシウムポンプの機能と構造変化を制御している52残基の膜タンパク質であるフォスフォランバンに関する分子動力学計算を行い、リン酸化前後の構造変化とそれによる制御機構を考察した。さらに、長時間のカルシウムポンプの分子運動を調べることにより、X線結晶構造解析で明らかになった大規模な構造変化とランダムな熱運動の関係を調べていくことが今後の課題である。

Over 30% of proteins are secreted across or integrated into membranes. Their newly synthesized forms contain either cleavable signal sequences or non-cleavable membrane anchor sequences, which direct them to the evolutionarily conserved Sec translocon (SecYEG in prokaryotes and Sec61, comprising a-, c- and bsubunits, in eukaryotes). The translocon then functions as a protein-conducting channel1. These processes of protein localization occur either at or after translation. In bacteria, the SecA ATPase2,3 drives post-translational translocation. The only high-resolution structure of a translocon available so far is that for SecYEb from the archaeonMethanococcus jannaschii4, which lacks SecA. Here we present the 3.2-Å -resolution crystal structure of the SecYE translocon from a SecA-containing organism, Thermus thermophilus. The structure, solved as a complex with an anti-SecY Fab fragment, revealed a ‘pre-open’ state of SecYE, in which several transmembrane helices are shifted, as compared to the previous SecYEb structure4, to create a hydrophobic crack open to the cytoplasm. Fab and SecA bind to a common site at the tip of the cytoplasmic domain of SecY.Molecular dynamics and disulphidemapping analyses suggest that the pre-open state might represent a SecYE conformational transition that is inducible by SecA binding. Moreover, we identified a SecA-SecYE interface that comprises SecA residues originally buried inside the protein, indicating that both the channel and the motor components of the Sec machinery undergo cooperative conformational changes on formation of the functional complex.

構造生物学の発展により、膜タンパク質などの重要な生命現象を担う生体分子の立体構造が次々に明らかになりつつある。分子動力学計算に代表される分子シミュレーションは、構造解析によって得られた立体構造を接続し、ダイナミックな描像を得るための研究手法として期待されている。しかし、タンパク質の機能する時間スケールと計算可能な時間スケールにはまだまだ大きなギャップが存在し、それを乗り越える手法の開発が必要である。これまでに開発された生体分子の構造探索手法を紹介し、今後の方向性を概観する。

Proper regulation of the intracellular ion concentration is essential to maintain life and is achieved by ion transporters that transport their substrates across the membrane in a strictly regulated manner. MgtE is a Mg²⁺ transporter that may function in the homeostasis of the intracellular Mg²⁺ concentration. A recent crystallographic study revealed that its cytosolic domain undergoes a Mg²⁺-dependent structural change, which is proposed to gate the ion-conducting pore passing through the transmembrane domain. However, the dynamics of Mg²⁺ sensing, i.e., how MgtE responds to the change in the intracellular Mg²⁺concentration, remained elusive. Here we performed molecular dynamics simulations of the MgtE cytosolic domain. The simulations successfully reproduced the structural changes of the cytosolic domain upon binding or releasingMg²⁺, as well as the ion selectivity. These results suggested the roles of the N and CBS domains in the cytosolic domain and their respective Mg²⁺ binding sites. Combined with the current crystal structures, we propose an atomically detailed model of Mg²⁺ sensing by MgtE.

Two new techniques are introduced in the generalized hybrid orbital (GHO) method [Pu , J. Phys. Chem. A 108, 632 (2004)] and tested on small molecules. The first is a way to determine occupation numbers dependent on the molecular mechanical (MM) atoms linked to the boundary. The method takes account of the inhomogeneity in the occupation numbers of the auxiliary orbitals from different types of MM atoms in such a way that the formal charge condition is fulfilled. The second technique is a rigorous orthogonalization procedure of auxiliary orbitals for more than two boundary atoms. It is shown that the new implementation widens the realm of the GHO method with flexible quantum mechanical/MM partitionings. (c) 2007 American Institute of Physics.

We examine the effects of the nanocrystal covalently bonded to one end, utilizing replica-exchange molecular dynamics simulation of a peptide with the sequence Ac-YGKAAAAKAAAAKAAAAKC-amide, to simulate the diffraction X-ray tracking (DXT) method. We performed three different simulations in this study. A simulation with no constraint, a simulation with one end fixed, and a simulation with one end fixed and also considering the effect of the nanocrystal by changing the mass of the sulfur atom in the C-terminus, which covalently bonded with the An nanocrystal in diffraction DXT method, was performed. The average configuration parameters of the three simulations are compared and discussed. We analyzed our simulation results utilizing principal component analysis. The obtained free-energy landscape indicated that the condition of the DXT technique will not affect the global-minimum state, however, it may affect the folding pathway.

We argue that the major driving force in protein folding is a gainin the water entropy. The formation of intramolecular hydrogen. bonds is important just for reducing the dehydration penalty as much as possible during the folding process. Focusing the physical basis on these two factors, we construct a new energy function which is calculated quite rapidly using our morphometric approach. Seven different proteins are chosen, and the native fold and over 600 misfolded structures are considered for each protein. It is shown that the energy function is always the lowest for the native structure.

G-peptide is a 16-residue peptide of the C-terminal end of streptococcal protein G B1 domain, which is known to fold into a specific β-hairpin within 6 μs. Here, we study molecular mechanism on the stability and folding of G-peptide by performing a, multicanonical. replica-exchange (MUCAREM) molecular dynamics simulation with explicit solvent. Unlike the preceding simulations of the same peptide, the simulation was started from an unfolded conformation without any experimental information on the native conformation. In the 278-ns trajectory, we observed three independent folding events. Thus MUCAREM can be estimated to accelerate the folding reaction more than 60 times than the conventional molecular dynamics simulations. The free-energy landscape of the peptide at room temperature shows that there are three essential subevents in the folding pathway to construct the native-like β-hairpin conformation: (i) a hydrophobic collapse of the peptide occurs with the side-chain contacts between Tyr45 and Phe52, (ii) then, the native-like turn is formed accompanying with the hydrogen-bonded network around the turn region, and (iii) finally, the rest of the backbone hydrogen bonds are formed. A number of stable native hydrogen bonds are formed cooperatively during the second stage, suggesting the importance of the formation of the specific turn structure. This is also supported by the accumulation of the nonnative conformations only with the hydrophobic cluster around Tyr45 and Phe52. These simulation results are consistent with high Phi-values of the turn region observed by experiment.

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