Ligand-dependent structural changes in the V1 ATPase from Manduca sexta

The response of V-1 ATPase of the tobacco hornworm Manduca 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 V-1 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 V-1 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 V-1 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.