Yohsuke Hagiwara

Functional Roles of a Structural Element Involving Na+-π Interactions in the Catalytic Site of T1 Lipase Revealed by Molecular Dynamics Simulations

Interactions between metal ions and pi systems (metal-pi interactions) are known to confer significant stabilization energy. However, in biological systems, few structures with metal-pi coordination have been determined; thus, its roles must still be elucidated. The cation-pi interactions are not correctly described by current molecular mechanics even when using a polarizable force field, and thus they require quantum mechanical calculations for accurate estimation. However, the huge computational costs of the latter methodologies prohibit long-time molecular dynamics (MD) simulations. Accordingly, we developed a novel scheme to obtain an effective potential for calculating the interaction energy with an accuracy comparable to that of advanced ab initio calculations at the CCSD(T) levels, and with computational costs comparable to those of conventional MM calculations. Then, to elucidate the functional roles of the Na(+)-phenylalanine (Phe) complex in the catalytic site of T1 lipase, we performed MD simulations in the presence/absence of the accurate Na(+)-pi interaction energy. A comparison of these MD simulations revealed that a significantly large enthalpy gain in Na(+)-Phe16 substantially stabilizes the catalytic site, whereas a water molecule could not be substituted for Na(+) for sufficient stabilization energy. Thus, the cation-pi interaction in the lipase establishes a remarkably stable core structure by combining a hydrophobic aromatic ring and hydrophilic residues, of which the latter form the catalytic triad, thereby contributing to large structural changes from the complex with ligands to the free form of the lipase. This is the first report to elucidate the detailed functional mechanisms of Na(+)-pi interactions.

Evaluation of stabilization energies in π–π and cation–π interactions involved in biological macromolecules by ab initio calculations

Non-covalent interactions involving aromatic rings contribute significantly to the stability of three-dimensional structures of biological macromolecules. Therefore, accurate descriptions of such interactions are crucial in understanding the functional mechanisms of biological molecules. However, it is also well known that, for some cases where van der Waals interactions make a dominant contribution, conventional ab initio electronic structure calculations, such as density functional theory, do not produce accurate interaction energies. In this study, we evaluated molecular mechanics (MM) calculations for two types of interactions involving aromatic rings, π-π interactions and cation-π interactions, by comparing our results with those obtained by advanced ab initio calculations at the coupled-cluster with singles, doubles and perturbative triples level. In structures with stacked aromatic rings, interaction energies obtained by MM calculations are overestimated. On the other hand, for cation-π interactions, the energies in MM calculations are significantly underestimated. In both cases, addition of an induction energy based on polarization effects also fails to improve the estimate given by MM calculations. The results indicate that current effective pairwise potentials are inappropriate to represent π-π and cation-π interactions.

Recent advances in jointed quantum mechanics and molecular mechanics calculations of biological macromolecules: schemes and applications coupled to ab initio calculations

We review the recent research on the functional mechanisms of biological macromolecules using theoretical methodologies coupled to ab initio quantum mechanical (QM) treatments of reaction centers in proteins and nucleic acids. Since in most cases such biological molecules are large, the computational costs of performing ab initio calculations for the entire structures are prohibitive. Instead, simulations that are jointed with molecular mechanics (MM) calculations are crucial to evaluate the long-range electrostatic interactions, which significantly affect the electronic structures of biological macromolecules. Thus, we focus our attention on the methodologies/schemes and applications of jointed QM/MM calculations, and discuss the critical issues to be elucidated in biological macromolecular systems.

Identification of the nucleophilic factors and the productive complex for the editing reaction by leucyl-tRNA synthetase

To ensure fidelity of translation, several aminoacyl‐tRNA synthetases (aaRSs) possess editing capability to hydrolyse mis‐aminoacylated tRNAs. In this report, based on our previously‐modelled structure of leucyl‐tRNA synthetase (LeuRS) complexed with valyl‐tRNALeu, further structural modelling has been performed along with molecular dynamics simulations. This enabled the identification of the nucleophile, which is different from that suggested by the crystal structure of the LeuRS • Nva2AA complex. Our results revealed that the 3′ hydroxyl group of A76 acts as a “gate” to regulate the accessibility of the nucleophile; thus, the opening of the gate leads to the productive complex for the reaction.

Structural modelling of the complex of leucyl-tRNA synthetase and mis-aminoacylated tRNALeu

To assure fidelity of translation, class Ia aminoacyl‐tRNA synthetases (aaRSs) edit mis‐aminoacylated tRNAs. Mis‐attached amino acids and structural water molecules are not included simultaneously in the current crystal structures of the aaRS•tRNA complexes, where the 3′‐ends (adenine 76; A76) are bound to the editing sites. A structural model of the completely solvated leucyl‐tRNA synthetase complexed with valyl‐tRNALeu was constructed by exploiting molecular dynamics simulations modified for the present modelling. The results showed that the ribose conformation of A76 is distinct from those observed in the above‐mentioned crystal structures, which could be derived from structural constraints in a sandwiched manner induced by the mis‐attached valine and tRNALeu.

Electronic and geometric structures of the blue copper site of azurin investigated by QM/MM hybrid calculations

The electronic and geometric structures of the copper-binding site in a fully solvated azurin were investigated using quantum mechanics (QM) and molecular mechanics (MM) hybrid calculations. Two types of computational models were applied to evaluate the effects of the environment surrounding the active site. In model I, long-distance electrostatic interactions between QM region atoms and partial point charges of the surrounding protein moieties and solvent water were calculated in a QM Hamiltonian, for which the spin-unrestricted Hartree-Fock (UHF)/density functional theory (DFT) hybrid all-electron calculation with the B3LYP functional was adopted. In model II, the QM Hamiltonian was not allowed to be polarized by those partial point charges. Models I and II provided different descriptions of the copper coordination structure, particularly for the coordinative bonds including a large dipole. In fact, the Cu-O(Gly45) and Cu-S(Cys112) bonds are sensitive to the treatment of long-distance electrostatic interactions in the QM Hamiltonian. This suggests that biological processes occurring in the active site are regulated by the surrounding structures of protein and solvent, and therefore the effects of long-range electrostatic interactions involved in the QM Hamiltonian are crucial for accurate descriptions of electronic structures of the copper active site of metalloenzymes.

Biological applications of hybrid quantum mechanics/molecular mechanics calculation.

Since in most cases biological macromolecular systems including solvent water molecules are remarkably large, the computational costs of performing ab initio calculations for the entire structures are prohibitive. Accordingly, QM calculations that are jointed with MM calculations are crucial to evaluate the long-range electrostatic interactions, which significantly affect the electronic structures of biological macromolecules. A UNIX-shell-based interface program connecting the quantum mechanics (QMs) and molecular mechanics (MMs) calculation engines, GAMESS and AMBER, was developed in our lab. The system was applied to a metalloenzyme, azurin, and PU.1-DNA complex; thereby, the significance of the environmental effects on the electronic structures of the site of interest was elucidated. Subsequently, hybrid QM/MM molecular dynamics (MD) simulation using the calculation system was employed for investigation of mechanisms of hydrolysis (editing reaction) in leucyl-tRNA synthetase complexed with the misaminoacylated tRNALeu, and a novel mechanism of the enzymatic reaction was revealed. Thus, our interface program can play a critical role as a powerful tool for state-of-the-art sophisticated hybrid ab initio QM/MM MD simulations of large systems, such as biological macromolecules.

Structural Instability of the Active Site of T1 Lipase Induced by Replacement of Na(+) with Water Complexed with the Phenylalanine Aromatic Ring.

The crystallographic analysis of T1 lipase suggested an interaction between Na(+) and the aromatic ring of Phe16 in the active site. However, experimental approaches could not dismiss the possible presence of water instead of Na(+). Our previous molecular dynamics (MD) simulations suggested that the significantly large enthalpy gain of the Na(+)-π interaction was required to preserve the catalytic core structure of T1 lipase. In this study, to examine the effects of water, we performed further MD simulations of T1 lipase involving the water-π interaction, instead of the Na(+)-π interaction, exploiting various force fields, such as ff99, ff02, and an accurate potential field to describe the water-π interaction, which was generated using our recently developed scheme (referred to as the grid-based energy representation). The analyses revealed that the water-π complex was unstable in the catalytic core of T1 lipase even when the accurate potential of the water-π complex represented by the grid-based energy function was employed in the MD simulations and led to the disruption of the coordinated structure. In contrast, the catalytic core structure of T1 lipase involving the Na(+)-π complex was significantly stable in the 10 ns MD simulation using the grid-based energy representation of the Na(+)-π interaction. Thus, the possible presence of water may be excluded, and our previous proposal concerning the functional role of the structural element involving the Na(+)-π interaction in the catalytic site of T1 lipase has unambiguously been confirmed. Further, the strong coordination of Na(+) and Nε of His358 was also shown to be substantial to preserve the core structure of the catalytic site.

Modulation of electronic structures of bases through DNA recognition of protein

The effects of environmental structures on the electronic states of functional regions in a fully solvated DNA·protein complex were investigated using combined ab initio quantum mechanics/molecular mechanics calculations. A complex of a transcriptional factor, PU.1, and the target DNA was used for the calculations. The effects of solvent on the energies of molecular orbitals (MOs) of some DNA bases strongly correlate with the magnitude of masking of the DNA bases from the solvent by the protein. In the complex, PU.1 causes a variation in the magnitude among DNA bases by means of directly recognizing the DNA bases through hydrogen bonds and inducing structural changes of the DNA structure from the canonical one. Thus, the strong correlation found in this study is the first evidence showing the close quantitative relationship between recognition modes of DNA bases and the energy levels of the corresponding MOs. Thus, it has been revealed that the electronic state of each base is highly regulated and organized by the DNA recognition of the protein. Other biological macromolecular systems can be expected to also possess similar modulation mechanisms, suggesting that this finding provides a novel basis for the understanding for the regulation functions of biological macromolecular systems.