SUBUNIT-EPSILON OF THE ESCHERICHIA-COLI ATP SYNTHASE - NOVEL INSIGHTSINTO STRUCTURE AND FUNCTION BY ANALYSIS OF 13 MUTANT FORMS

Structural models of subunit epsilon of the ATP synthase from Escheric hia coli have been determined recently by NMR [Wilkens et al. (1995) N at. Struct. Biol. 2, 961-967] and by X-ray crystallography [Uhlin et a l. (1997) Structure 5, 1219-1230], revealing a two-domain protein. In this study, six new epsilon mutants were constructed and analyzed: Y63 A, D81A, T82A, and three truncated mutants, tr80(S), tr94-(LAS), and t r117(AS). Seven mutants constructed previously were also analyzed: E31 A, E59A, S65A, E70A, T77A, R58A, and D81A/R85A. Subunits were purified by isoelectric focusing from extracts of cells that overproduced thes e 13 mutants. F-1 was prepared lacking subunit epsilon by immobilized- Ni affinity chromatography. Three mutants, E70A, S65A, and E31A, showe d somewhat higher affinities and extents of inhibition than the wild t ype. Three mutants, T82A, R85A, and tr94(LAS), showed both lower affin ities and extents of inhibition, over the concentration range tested. Two showed no inhibition, D81A and tr80(S). The others, T77A, Y63A, E5 9A, and tr117(AS), showed lower affinities than wild type, but the ext ents of inhibition were nearly normal. Results indicate that the C-ter minal domain of subunit epsilon contributes to inhibition of ATP hydro lysis, but it is not necessary for ATP-driven proton translocation. In teractions with subunit gamma are likely to involve a surface containi ng residues S65, E70, T77, D81, and T82, while residues R85 and Y63 ar e likely to be important in the conformation of subunit epsilon.