Theoretical studies of the ATP hydrolysis mechanism of myosin

ABSTRACT The ATP hydrolysis mechanism of myosin was studied using quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the energy-minimized structure of the myosin(Sl dc)-ATP complex, which was determined by molecular mechanics calculations. The result of QM calculations suggested that the ATP hydrolysis mechanism of myosin consists of a single elementary reaction in which a water molecule nucleophilically attacked gamma-phosphorus of ATP. In addition, we performed molecular dynamics simulations of the initial and final states of the ATP hydrolysis reaction, that is, the myosin-ATP and myosin-ADP-Pi complexes. These calculations revealed roles of several amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the ATPase pocket. Lys185 maintains the conformation of Beta and gamma-phosphate groups of ATP by forming the hydrogen bonds. Thrl 86 and Ser237 are coordinated to a Mg^sup 2^+ ion, which interacts with the phosphates of ATP and therefore contributes to the stabilization of the ATP structure. Arg238 and Glu459, which consisted of the gate of the ATPase pocket, retain the water molecule acting on the hydrolysis at the appropriate position for initiating the hydrolysis.

INTRODUCTION

Myosins are molecular motors that track along actin filaments through the hydrolysis of ATP and play an important role in diverse biological contractile events. Recently, the three-dimensional structures of myosins have been determined by x-ray crystallography (Rayment et al., 1993; Fisher et al., 1995; Smith and Rayment, 1996; Gulick et al., 1997). These structural data indicated that nucleotide (ATP or ADP)- and actin-binding sites were located in the globular head of myosin, called subfragment 1 or SI.

In Dictyostelium myosin II (Sldc), the nucleotide-binding site (called the ATPase pocket) is surrounded by three loop structures; the P-loop (residues 179-186), the switch I loop (residues 233-240), and the switch II loop (residues 454-459). The ATPase pocket has a gate for release of phosphate, a product of ATP hydrolysis. This gate is closed or opened with the formation or disappearance of ionic bonds between the side chains of Arg238 (in the switch I loop) and Glu459 (in the switch II loop). Determination of the crystal structures of Sldc with several nucleotides and its analogs (Fisher et al., 1995; Smith and Rayment., 1996; Gulick et al., 1997) revealed that the switch I loop undergoes a minor conformational change and that the switch II loop undergoes a large conformational change. In the structures of Sldc with MgADP/BeFx, MgAMPPNP, MgATP-gammaS, or MgADP, the switch II loop moved away from the ATPase pocket, so that the gate was opened with the disappearance of ionic bonds. In contrast, the structures of the Sldc with MgADP/VO4 or MgADP/AIFx indicated that Arg238 formed ionic bonds with Glu459 and, consequently, the gate was closed. From analysis of these crystal structures, Rayment et al. (1993) suggested that the conformation observed in the structure of S I dc with MgADP/V04 (Fisher et al., 1995), where the gate was closed, was necessary for the hydrolysis of ATP. In addition, many experiments on site-directed mutation of myosin have suggested that several amino acid residues of the ATPase pocket play an important role in the hydrolysis of ATP (Sasaki and Sutoh, 1998; Li et al., 1998; Furch et al., 1999; Onishi et al., 1998). From these studies, it was revealed that Lys185, Arg238, and Glu.459 were closely related with the hydrolysis of ATP. It is also known that ionic bonds between Arg238 (in the switch I loop) and Glu459 (in the switch II loop) are required to support efficient ATP hydrolysis.

According to the suggestion by Bagshaw et al. (1974), the mechanism of the binding and hydrolysis of ATP by myosin consists of seven steps as shown in scheme 1, where M is myosin and Pi is a phosphate. The asterisks refer to different conformational states as detected by intrinsic protein fluorescence.

This work was supported in part by the super computer Vpp700e in Institute of Physical and Chemical Research (RIKEN). The authors thank the Research Center for Computational Science, Okazaki. The computations were also carried out by the DRIA System at the Faculty of Pharmaceutical Sciences, Chiba University.

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[Author Affiliation]

Noriaki Okimoto,* Kazunori Yamanaka,^ Junko Ueno,t Masayuki Hata,^ Tyuji Hoshino,^ and Minoru Tsuda^

[Author Affiliation]

*Computational Science Laboratory, Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, 351-0198, Japan and

^Laboratory of Physical Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan

[Author Affiliation]

Received for publication 29 September 2000 and in final form 31 July 2001.

Address reprint requests to Dr. Noriaki Okiomoto, Institute of Physical and Chemical Research (RIKEN), Computational Science Laboratory, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. Tel.: 81-48-467-9417; Fax: 81-48-467-4078; E-mail: okimoto@atlas.riken.go.jp.