Mutant LV476-7AA of A-subunit of Enterococcus hirae V1-ATPase: High affinity of A3B3 complex to DF axis and low ATPase activity

• Published in: SpringerPlus Vol. 2; no. 1; p. 689
• Main Authors: Alam, Md Jahangir, Yamato, Ichiro, Arai, Satoshi, Saijo, Shinya, Mizutani, Kenji, Ishizuka-Katsura, Yoshiko, Ohsawa, Noboru, Terada, Takaho, Shirouzu, Mikako, Yokoyama, Shigeyuki, Iwata, So, Kakinuma, Yoshimi, Murata, Takeshi
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 27.12.2013; BioMed Central Ltd
• Subjects: Biomedical and Life Sciences; Humanities and Social Sciences; multidisciplinary; Science; Science (multidisciplinary); Site-directed mutation; Catalytic domain; Reconstitution; Surface plasmon resonance; ATPase assay
• ISSN: 2193-1801; 2193-1801
• DOI: 10.1186/2193-1801-2-689

Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble functional domain V 1 (A 3 B 3 DF) and an integral membrane domain V o (ac), where V 1 and V o 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 A 3 B 3 heterohexamer with D-subunit in DF heterodimer in the crystal structures of A 3 B 3 and A 3 B 3 DF. In our previous study, we reported 10 mutants of E. hirae V 1 -ATPase, which showed lower binding affinities of DF with A 3 B 3 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 A 3 B 3 complex with higher binding affinities for wild-type or mutant DF heterodimers and lower initial ATPase activities compared to the wild-type A 3 B 3 complex, consistent with our previous proposal of reciprocal relationship between the ATPase activity and the protein-protein binding affinity of DF axis to the A 3 B 3 catalytic domain of E. hirae V-ATPase. These observations suggest that the binding of DF axis at the contact region of A 3 B 3 rotary ring is relevant to its rotation activity.