The Torque of Rotary F-ATPase Can Unfold Subunit Gamma If Rotor and Stator Are Cross-Linked

Abstract

During ATP hydrolysis by F-1-ATPase subunit gamma rotates in a hydrophobic bearing, formed by the N-terminal ends of the stator subunits (alpha beta)(3). If the penultimate residue at the alpha-helical C-terminal end of subunit gamma is artificially cross-linked (via an engineered disulfide bridge) with the bearing, the rotary function of F-1 persists. This observation has been tentatively interpreted by the unfolding of the alpha-helix and swiveling rotation in some dihedral angles between lower residues. Here, we screened the domain between rotor and bearing where an artificial disulfide bridge did not impair the rotary ATPase activity. We newly engineered three mutants with double cysteines farther away from the C-terminus of subunit gamma, while the results of three further mutants were published before. We found ATPase and rotary activity for mutants with cross-links in the single alpha-helical, C-terminal portion of subunit gamma (from gamma 285 to gamma 276 in E. coli), and virtually no activity when the cross-link was placed farther down, where the C-terminal alpha-helix meets its N-terminal counterpart to form a supposedly stable coiled coil. In conclusion, only the C-terminal singular alpha-helix is prone to unwinding and can form a swivel joint, whereas the coiled coil portion seems to resist the enzyme's torque.