Molecular contacts in the transmembrane c-subunit oligomer of F-ATPases identified by tryptophan substitution mutagenesis

When isolated in its monomeric form, subunit c of the proton transporting A TP synthase of Escherichia coli was shown to fold in a hairpin-like structu re consisting of two hydrophobic membrane spanning helices and a short conn ecting hydrophilic loop. In the plasma membrane of Escherichia coli, howeve r, about 9-12 c-subunit monomers form an oligomeric complex that functions in transmembrane proton conduction and in energy transduction to the cataly tic F-1 domain. The arrangement of the monomers and the molecular architect ure of the complex were studied by tryptophan scanning mutagenesis and rest rained MD simulations. Residues 12-24 of the N-terminal transmembrane segme nt of subunit c were individually substituted by the large and moderately h ydrophobic tryptophan side chain. Effects on the activity of the mutant pro teins were studied in selective growth experiments and various ATP synthase specific activity assays. The results identify potential intersubunit cont acts and structurally non-distorted, accessible residues in the c-oligomer and add constraints to the arrangement of monomers in the oligomeric comple x. Results from our mutagenesis experiments were interpreted in structural models of the c-oligomer that have been obtained by restrained MD simulatio ns. Different stoichiometries and monomer orientations were applied in thes e calculations. A cylindrical complex consisting of 10 monomers that are ar ranged in two concentric rings with the N-terminal helices of the monomers located at the periphery shows the best match with the experimental data. ( C) 2000 Elsevier Science B.V. All rights reserved.