Eukaryotic vacuolar ATPase (V-ATPase) is regulated by a reversible dissociation mechanism that involves breaking and reforming of protein-protein interactions at the interface of the V(1)-ATPase and V(o)-proton channel domains. We found previously that the head domain of the single copy C subunit (C(head)) binds one subunit EG heterodimer with high affinity (Oot, R.A. and Wilkens, S. (2010) J. Biol. Chem. 285, 24654-24664). Here we generated a water-soluble construct of the N-terminal domain of the V(o) "a" subunit composed of amino acid residues 104-372 (a(NT(104-372))). Analytical gel filtration chromatography and sedimentation velocity analysis revealed that a(NT(104-372)) undergoes reversible dimerization in a concentration-dependent manner. A low-resolution molecular envelope was calculated for the a(NT(104-372)) dimer using small angle x-ray scattering data. Isothermal titration calorimetry experiments revealed that a(NT(104-372)) binds the C(foot) and EG heterodimer with dissociation constants of 22 and 33 μM, respectively. We speculate that the spatial closeness of the a(NT), C(foot), and EG binding sites in the intact V-ATPase results in a high-avidity interaction that is able to resist the torque of rotational catalysis, and that reversible enzyme dissociation is initiated by breaking either the a(NT(104-372))-C(foot) or a(NT(104-372))-EG interaction by an as-yet unknown signaling mechanism.

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Journal Article | Research Support, N.I.H., Extramural | Research Support, U.S. Gov't, Non-P.H.S.

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Oot RA, Wilkens S

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