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| Title | Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel: DEFINITION OF THE INTERACTION OF ROTOR AND STATOR AND IMPLICATIONS FOR ENZYME REGULATION BY REVERSIBLE DISSOCIATION. |
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| Journal, issue, pages | J Biol Chem, Vol. 292, Issue 5, Page 1749-1761, Year 2017 |
| Publish date | Feb 3, 2017 |
 Authors | Nicholas J Stam / Stephan Wilkens /  |
| PubMed Abstract | Eukaryotic vacuolar H-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V-ATPase and membrane-integral ...Eukaryotic vacuolar H-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V-ATPase and membrane-integral V proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast V The structure indicated that, when V is released from V, the N-terminal cytoplasmic domain of subunit a (a) changes conformation to bind rotor subunit d However, insufficient resolution precluded a precise definition of the a-d interface. Here we reconstituted V into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between a and d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the a-d interaction, as shown by isothermal titration calorimetry and gel filtration of recombinant subunits. A recent cryo-EM study of holo V-ATPase revealed three major conformations corresponding to three rotational states of the central rotor of the enzyme. Comparison of the three V-ATPase conformations with the structure of nanodisc-bound V revealed that V is halted in rotational state 3. Combined with our prior work that showed autoinhibited V-ATPase to be arrested in state 2, we propose a model in which the conformational mismatch between free V and V functions to prevent unintended reassembly of holo V-ATPase when activity is not needed. |
 External links | J Biol Chem / PubMed:27965356 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 16.3 - 20.3 Å |
| Structure data |  EMDB-6335:  EMDB-6336: |
| Source |
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Saccharomyces cerevisiae (brewer's yeast)
