Essential role of the conformational flexibility of helices 1 and 5 on thelipid binding activity of apolipophorin-III

It has been recently postulated that the conformational flexibility of heli ces 1 and 5 of Locusta migratoria apoLp-III could play an important role in early steps of binding of this apolipoprotein to a lipid surface (Soulages , J. L., and Arrese, E. L. (2000) J. Biol. Chem. 275, 17501-17509). To test this model, we have designed a double Cys mutant in which a disulfide bond linking helices 1 and 5 could be formed, resulting in an apolipoprotein wi th reduced conformational flexibility of its N- and C-terminal helices. Sub stitution of Thr(18) and Ala(147) by Cys residues provided a protein that u nder nonreducing conditions was fully oxidized. The far-UV CD spectra of th is mutant in the reduced and oxidized states indicated that their secondary structures were identical to the structure of the wild type recombinant ap oLp-III, which contains no Cys residues. Near-UV CD studies confirmed the f ormation of a disulfide bond and the absence of structural perturbations. T he lipid binding activity of the reduced mutant, as determined by its abili ty to form discoidal lipoproteins, was nearly identical to that of the wild type protein. Contrarily, the disulfide form of the mutant was not able to form discoidal lipoproteins with liposomes of either dimirystoylphosphatid ylcholine or dimyristoylphosphatidylglycerol. It is concluded that the sepa ration of the helices 1 and 5 constitutes one of the key steps along the co mplex pathway for the formation of the final apolipoprotein lipid-bound sta te. It is inferred that the conformational flexibility of helices 1 and 5 i s a key property of apoLp-III, allowing the exposure of hydrophobic protein regions and the interaction of the hydrophobic faces of the amphipathic al pha -helices with the lipoprotein lipid surface.