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

Md Jahangir Alam,Yoshimi Kakinuma,Ichiro Yamato,Takeshi Murata,Kenji Mizutani,Noboru Ohsawa,Mikako Shirouzu,Shigeyuki Yokoyama,So Iwata,Shinya Saijo,Satoshi Arai,Yoshiko Ishizuka-Katsura,Takaho Terada

doi:10.1186/2193-1801-2-689

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 (LV476-7AA) 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.

Highlights

Vacuolar ATPase (V-ATPase) functions as a proton pump in the acidic organelles and plasma membranes of eukaryotic cells and bacteria (Forgac 2007; Stevens & Forgac 1997)
Reconstitution and purification of A(LV476-7AA)3B3 and A(LV476-7AA)3B(L389A)3 heterohexamers From the crystal structures of A3B3 and A3B3DF (Arai et al 2013), we identified LV476-7 of A-subunit and L389 of B-subunit located closely with the interacting Dsubunit in the “tight” form (ACR-BCR pair) of V1-ATPase (Figure 1C)
These complexes seemed stable in the absence of nucleotides, A(LV476-7AA) 3B3 and A(LV476-7AA)3B(L389A)3 heterohexamers showed lower stability than the wild-type A3B3; in native PAGE, a band at position of A(LV476-7AA)1B1 or A(LV476-7AA)1B (L389A)1 complex was observed after a few days storage at −80 ̊C and A(LV476-7AA)3B(L389A)3 heterohexamer dissociated into monomers after a few weeks storage at 4°C (Figure 2A, lane 4)

Summary

Introduction

Vacuolar ATPase (V-ATPase) functions as a proton pump in the acidic organelles and plasma membranes of eukaryotic cells and bacteria (Forgac 2007; Stevens & Forgac 1997). During ATP hydrolysis, the central axis (the DFd complex) attached on the membrane c-ring rotates inside the hexagonally arranged A3B3 complex, which causes ion pumping at the interface between the c-ring and a-subunit (Murata et al 2008). We reported the reconstitution and purification of A3B3 and A3B3DF of E. hirae V-ATPase (Arai et al 2009) and solved the crystal structures of DF, A3B3, and A3B3DF (Arai et al 2013; Saijo et al 2011) Crystal structures of these complexes suggest the existence of 120 polar and nonpolar (van der Waals) interactions between the A3B3 and DF complexes and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue (Arai et al 2013). We reported the mutational effects of 10 interacting residues at the conserved C-terminal domain (near the 480DSLSDND486 sequence of A-subunit (Figure 1F), probably corresponding to the DELSEED sequence of F-ATPase (Mnatsakanyan et al 2011; Nakanishi-Matsui & Futai 2008)) of A- and B-subunits with the residues of D-subunit, showing higher initial

Methods

Results

Conclusion