Abstract
Vacuolar ATPases (V-ATPases) function as proton pumps in various cellular membrane systems. The hydrophilic V1 portion of the V-ATPase is a rotary motor, in which a central-axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex by using ATP hydrolysis energy. We have previously reported crystal structures of Enterococcus hirae V-ATPase A3B3 and A3B3DF (V1) complexes; the result suggested that the DF axis induces structural changes in the A3B3 complex through extensive protein-protein interactions. In this study, we mutated 10 residues at the interface between A3B3 and DF complexes and examined the ATPase activities of the mutated V1 complexes as well as the binding affinities between the mutated A3B3 and DF complexes. Surprisingly, several V1 mutants showed higher initial ATPase activities than wild-type V1-ATPase, whereas these mutated A3B3 and DF complexes showed decreased binding affinities for each other. However, the high ATP hydrolysis activities of the mutants decreased faster over time than the activity of the wild-type V1 complex, suggesting that the mutants were unstable in the reaction because the mutant A3B3 and DF complexes bound each other more weakly. These findings suggest that strong interaction between the DF complex and A3B3 complex lowers ATPase activity, but also that the tight binding is responsible for the stable ATPase activity of the complex.
Highlights
Vacuolar ATPase (V-ATPase) functions as a proton pump in acidic organelles and plasma membranes of eukaryotic cells and bacteria [1,2]
ATP hydrolysis induces the rotation of the central axis (DFd complex) and the attached membrane c ring, which results in ions being pumped through a channel at the interface between the c ring and the a subunit
We purified the A3B3 and DF complexes and reconstituted the V1-ATPase using the 2 complexes [14] and we determined the crystal structures of the DF, A3B3, and A3B3DF complexes [15,16]. This structural information suggests that the DF complex binds the A3B3 complex tightly through 19 polar interactions and 101 nonpolar interactions; through these interactions, the DF complex induces conformational changes in the A3B3 complex (Figure 2C-H), and ATP hydrolysis appears to be stimulated by the approach of a conserved arginine residue [15]
Summary
Vacuolar ATPase (V-ATPase) functions as a proton pump in acidic organelles and plasma membranes of eukaryotic cells and bacteria [1,2]. We reconstituted mutant V1-domains, and measured the ATPase activities and binding affinities of the mutant A3B3 for the DF axis (wild-type or mutant DF). Slightly lower initial ATPase activity and slightly higher binding affinity than the wild-type DF (Table 1).
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