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[摘要]：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 mu M, 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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