Ligand-Dependent Structural Changes in the V1 ATPase from Manduca sexta

The response of V1 ATPase of the tobacco hornwormManduca sexta to Mg2+ and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl2-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V1 complex was supplemented with CuCl2 nucleotide-dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP·PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V1 complex. The catalytic subunit A was reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP·PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V1 ATPase from M. sexta and conformational alterations in this enzyme due to Mg2+ and nucleotide binding are discussed on the basis of these and previous observations.