Engineering of IF 1 -susceptive bacterial F 1 -ATPase.

IF 1 , an inhibitor protein of mitochondrial ATP synthase, suppresses ATP hydrolytic activity of F 1 . One of the unique features of IF 1 is the selective inhibition in mitochondrial F 1 (MF 1 ); it inhibits catalysis of MF 1 but does not affect F 1 with bacterial origin despite high sequence homology between MF 1 and bacterial F 1 . Here, we aimed to engineer thermophilic Bacillus F 1 (TF 1 ) to confer the susceptibility to IF 1 for elucidating the molecular mechanism of selective inhibition of IF 1 . We first examined the IF 1 -susceptibility of hybrid F 1 s, composed of each subunit originating from bovine MF 1 (bMF 1 ) or TF 1 . It was clearly shown that only the hybrid with the β subunit of mitochondrial origin has the IF 1 -susceptibility. Based on structural analysis and sequence alignment of bMF 1 and TF 1 , the five non-conserved residues on the C-terminus of the β subunit were identified as the candidate responsible for the IF 1 -susceptibility. These residues in TF 1 were substituted with the bMF 1 residues. The resultant mutant TF 1 showed evident IF 1 -susceptibility. Reversely, we examined the bMF 1 mutant with TF 1 residues at the corresponding sites, which showed significant suppression of IF 1 -susceptibility, confirming the critical role of these residues. We also tested additional three substitutions with bMF 1 residues in α and γ subunits that further enhanced the IF 1 -susceptibility, suggesting the additive role of these residues. We discuss the molecular mechanism by which IF 1 specifically recognizes F 1 with mitochondrial origin, based on the present result and the structure of F 1 -IF 1 complex. These findings would help the development of the inhibitors targeting bacterial F 1 .