OLIGOMYCIN SENSITIVITY CONFERRING PROTEIN (OSCP) OF BOVINE HEART MITOCHONDRIAL ATP SYNTHASE - HIGH-AFFINITY OSCP-F-0 INTERACTIONS REQUIRE ALOCAL ALPHA-HELIX AT THE C-TERMINAL END OF THE SUBUNIT

Earlier studies on oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase (F1Fo) indicated that a deletion mu tant form (CD-10), lacking the last 10 amino acid residues (K181-L190) , was unable to bind to the F-o segment, or reconstitute energy-linked reactions in OSCP-depleted F1Fo complexes [Joshi et al. (1996) Bioche mistry 35, 12094-12103]. So far as known, the K181-L190 region of all mammalian species of OSCP harbors four charged residues at positions 1 81, 184, 187, and 188, while secondary structure predictions suggest t hat the K178-M186 region has a high propensity to form a helix [Ovchin nikov et al, (1984) FEES Lett. 166, 19-22; Higuti et al, (1993) Biochi m. 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 re placements of all four charged residues by uncharged but polar glutami nes, 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 co mplete loss in the coupling activity of the resultant mutant. A detail ed analysis of the 6-proline mutant form revealed that the variant was indistinguishable from WT OSCP with respect to expression characteris tics, affinity for S-Sepharose, and ability to interact with F-1, but was unable to complex with the F-o segment. These studies suggest that the global protein structure was not destabilized. The helix potentia l prediction analyses showed that the 6-proline OSCP displayed a marke d decrease in the helix-forming propensity in the region corresponding to residues 175-190, Quantitative CD analyses to measure helical cont ent demonstrated that both of the mutant forms 6-proline-OSCP and CD-1 0 had a somewhat lower alpha-helical content compared to WT protein, w hile synthetic peptides corresponding in sequence to the K178-Ll90 reg ion displayed a high propensity to form a helix. Tal;en together, thes e results suggest that the C-terminal end of OSCP encompasses an alpha -helix which is crucial for high-affinity interactions of the C-termin al end of this subunit with F-o in the F1Fo enzyme.