Three intragenic second-site suppressors, P353L, T237I, and L390F, were identified that suppressed two mutations in, and one adjacent to, the P-loop in the -subunit of the yeast F-ATPase. The crystal structure of bovine F-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E.(1994) Nature 370, 621-628) shows that these suppressor residues are located in the nucleotide-binding domain. Specific hypotheses have been formulated that suggest the conformational coupling of the P-loop with the suppressor sites. P353L is in a ``catch'' region, which forms unique interactions with the -subunit in the three different conformational states of the catalytic site. The identification of this suppressor mutation demonstrates genetically that the catch region is conformationally coupled to the P-loop. T237I is shown to interact with Lys-209, which occurs just after the P-loop. This suggests that this interaction changes the conformation of the P-loop to suppress the initial mutation. L390F interacts with Ala-181, which is adjacent to the P-loop. The mechanism of this suppression is suggested to occur through the interactions of L390F with Ala-181. These results identify critical interactions that modulate the structure of the P-loop and thus the biochemistry of the enzyme.

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