Engineering of IF -susceptive bacterial F -ATPase.

IF , an inhibitor protein of mitochondrial ATP synthase, suppresses ATP hydrolytic activity of F . One of the unique features of IF is the selective inhibition in mitochondrial F (MF ); it inhibits catalysis of MF but does not affect F with bacterial origin despite high sequence homology between MF and bacterial F . Here, we aimed to engineer thermophilic Bacillus F (TF ) to confer the susceptibility to IF for elucidating the molecular mechanism of selective inhibition of IF . We first examined the IF -susceptibility of hybrid F s, composed of each subunit originating from bovine MF (bMF ) or TF . It was clearly shown that only the hybrid with the β subunit of mitochondrial origin has the IF -susceptibility. Based on structural analysis and sequence alignment of bMF and TF , the five non-conserved residues on the C-terminus of the β subunit were identified as the candidate responsible for the IF -susceptibility. These residues in TF were substituted with the bMF residues. The resultant mutant TF showed evident IF -susceptibility. Reversely, we examined the bMF mutant with TF residues at the corresponding sites, which showed significant suppression of IF -susceptibility, confirming the critical role of these residues. We also tested additional three substitutions with bMF residues in α and γ subunits that further enhanced the IF -susceptibility, suggesting the additive role of these residues. We discuss the molecular mechanism by which IF specifically recognizes F with mitochondrial origin, based on the present result and the structure of F -IF complex. These findings would help the development of the inhibitors targeting bacterial F .