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Abstract:
The three-dimensional structure of the Manduca sexta midgut V1 ATPase has been determined at 3.2 nm resolution from electron micrographs of negatively stained specimens. The V1 complex has a barrel-like structure 11 nm in height and 13.5 nm in diameter. It is hexagonal in the top view, whereas in the side view, the six large subunits A and B are interdigitated for most of their length (9 nm). The topology and importance of the individual subunits of the V1 complex have been explored by protease digestion, resistance to chaotropic agents, MALDI-TOF mass spectrometry, and CuCl2-induced disulfide formation. Treatment of V1 with trypsin or chaotropic iodide resulted in a rapid cleavage or release of subunit D from the enzyme, indicating that this subunit is exposed in the complex. Trypsin cleavage of V1 decreased the ATPase activity with a time course that was in line with the cleavage of subunits B, C, G, and F. When CuCl2 was added to V1 in the presence of CaADP, the cross-linked products A−E−F and B−H were generated. In experiments where CuCl2 was added after preincubation of CaATP, the cross-linked products E−F and E−G were formed. These changes in cross-linking of subunit E to near-neighbor subunits support the hypothesis that these are nucleotide-dependent conformational changes of the E subunit.
