title: Mutant LV(476-7)AA of A-subunit of Enterococcus hirae V1-ATPase: High affinity of A3B3 complex to DF axis and low ATPase activity.
abstract: Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble functional domain V1 (A3B3DF) and an integral membrane domain Vo (ac), where V1 and Vo domains are connected by a central stalk, composed of D-, F-, and d-subunits; and two peripheral stalks (E- and G-subunits). We identified 120 interacting residues of A3B3 heterohexamer with D-subunit in DF heterodimer in the crystal structures of A3B3 and A3B3DF. In our previous study, we reported 10 mutants of E. hirae V1-ATPase, which showed lower binding affinities of DF with A3B3 complex leading to higher initial specific ATPase activities compared to the wild-type. In this study, we identified a mutation of A-subunit (LV(476-7)AA) at its C-terminal domain resulting in the A3B3 complex with higher binding affinities for wild-type or mutant DF heterodimers and lower initial ATPase activities compared to the wild-type A3B3 complex, consistent with our previous proposal of reciprocal relationship between the ATPase activity and the protein-protein binding affinity of DF axis to the A3B3 catalytic domain of E. hirae V-ATPase. These observations suggest that the binding of DF axis at the contact region of A3B3 rotary ring is relevant to its rotation activity.
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585 Japan ; Department of Genetic Engineering and Biotechnology, School of Life Sciences, Shahjalal University of Science and Technology, Sylhet, 3114 Bangladesh.
authors.forename: Jahangir
authors.initials: J
authors.lastname: Alam
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585 Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Yamato
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585 Japan ; Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-choInage, Chiba, 263-8522 Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Arai
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585 Japan ; RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo, 679-5148 Japan ; Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki, 305-0801 Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Saijo
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585 Japan ; Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-choInage, Chiba, 263-8522 Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Mizutani
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Ishizuka-Katsura
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Ohsawa
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan ; RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan.
authors.forename: Takaho
authors.initials: T
authors.lastname: Terada
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan ; RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan ; Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, 606-8501 Japan.
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.affiliation: Laboratory of Molecular Physiology and Genetics, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan.
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Kakinuma
authors.affiliation: Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-choInage, Chiba, 263-8522 Japan ; RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan ; JST, PRESTO, 1-33 Yayoi-cho, Inage, Chiba, 263-8522 Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
keywords: ATPase assay; Catalytic domain; Enterococcus hirae; Reconstitution; Site-directed mutation; Surface plasmon resonance