Oligomycin Sensitivity Conferring Protein (OSCP) of Bovine Heart Mitochondrial ATP Synthase:  High-Affinity OSCP−F_o Interactions Require a Local α-Helix at the C-Terminal End of the Subunit

Abstract

Earlier studies on oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase (F1Fo) indicated that a deletion mutant form (CD-10), lacking the last 10 amino acid residues (K181-L190), was unable to bind to the Fo segment, or reconstitute energy-linked reactions in OSCP-depleted F1Fo complexes [Joshi et al. (1996) Biochemistry 35, 12094-12103]. So far as known, the K181-L190 region of all mammalian species of OSCP harbors four charged residues at positions 181, 184, 187, and 188, while secondary structure predictions suggest that the K178-M186 region has a high propensity to form a helix [Ovchinnikov et al. (1984) FEBS Lett. 166, 19-22; Higuti et al. (1993) Biochim. Biophys. Acta 1172, 311-314; Grinkevich et al. ( 1994) Biol. Membr. 11, 310-323; Engelbrecht et al. (1991) Z. Naturforsch., C: Biochem., Biophys., Biol.,Virol. 46, 759-764]. Present studies were undertaken to clarify the role of individual amino acids in the K181-L190 region in OSCP-stimulated energy coupling. Our data show that simultaneous replacements of all four charged residues by uncharged but polar glutamines, or of K181-R184 by apolar alanines, had no significant influence either on the total alpha-helix content of the mutant forms or on the ability of mutant OSCPs to couple energy-linked reactions. However, a substitution of the K181-M186 region by six proline residues led to complete loss in the coupling activity of the resultant mutant. A detailed analysis of the 6-proline mutant form revealed that the variant was indistinguishable from WT OSCP with respect to expression characteristics, affinity for S-Sepharose, and ability to interact with F1, but was unable to complex with the Fo segment. These studies suggest that the global protein structure was not destabilized. The helix potential prediction analyses showed that the 6-proline OSCP displayed a marked decrease in the helix-forming propensity in the region corresponding to residues 175-190. Quantitative CD analyses to measure helical content demonstrated that both of the mutant forms 6-proline-OSCP and CD-10 had a somewhat lower alpha-helical content compared to WT protein, while synthetic peptides corresponding in sequence to the K178-L190 region displayed a high propensity to form a helix. Taken together, these results suggest that the C-terminal end of OSCP encompasses an alpha-helix which is crucial for high-affinity interactions of the C-terminal end of this subunit with Fo in the F1Fo enzyme.

The b and δ Subunits of the Escherichia coli ATP Synthase Interact via Residues in their C-terminal Regions

“…Together these subunits are believed to form a "second stalk" reaching from the membrane to near the top of the F 1 sector, which may function to hold the ␣ 3 ␤ 3 subunits stationary during rotation of ␥ and ⑀ driven by proton flow through F 0 (48,49). Earlier truncation and mutagenesis studies showed that the C terminus of b was essential for proper assembly of ATP synthase (10) and implied that the C terminus of ␦ played a role in interaction of F 1 with the F 0 sector (21)(22)(23)(24).…”

“…Membranes of mutant E. coli strains expressing truncated forms of ␦ showed little ATPase activity (21), suggesting that F 1 cannot bind to the membrane in the absence of ␦. In truncation and mutagenesis studies using the mitochondrial (22,23) and yeast (24) homologues of ␦, called OSCP 1 for oligomycin sensitivity conferring protein, the C-terminal region of OSCP was implicated in F 0 binding.…”

The Oligomycin-Sensitivity Conferring Protein of Mitochondrial ATP Synthase: Emerging New Roles in Mitochondrial Pathophysiology

“…, in the coupled enzyme [70]. In this context, depletion/reconstitution studies in isolated membranes demonstrated that OSCP is absolutely necessary in coupling proton translocation to ATP synthesis [71]. In keeping with this key function of the subunit, yeast OSCP knockouts failed to survive under normal growth conditions [73].…”

The structure of the membrane extrinsic region of bovine ATP synthase

“…Fragments of subunit b and the OSCP with intact C-terminal regions form somewhat unstable heterodimeric complexes that are stabilized by F 6 . Deletion of residues 185-214 of subunit b prevents any association between the OSCP and the rest of the stator (6), and deletion of the C-terminal region of the yeast OSCP uncouples pmf partially from ATP synthesis (14). Also, biotin attached to the C-terminus of the yeast OSCP was detected at the junction between the crown and the nucleotide binding domains of F 1 (15), which is consistent with the position of the C-terminal region of the OSCP in the bovine F 1 -stator T complex.…”