Essential role of the conformational flexibility of helices 1 and 5 on the lipid binding activity of apolipophorin-III.
It has been recently postulated that the conformational flexibility of helices 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 with reduced conformational flexibility of its N- and C-terminal helices. Substitution of Thr(18) and Ala(147) by Cys residues provided a protein that under nonreducing conditions was fully oxidized. The far-UV CD spectra of this mutant in the reduced and oxidized states indicated that their secondary structures were identical to the structure of the wild type recombinant apoLp-III, which contains no Cys residues. Near-UV CD studies confirmed the formation of a disulfide bond and the absence of structural perturbations. The lipid binding activity of the reduced mutant, as determined by its ability 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 dimirystoylphosphatidylcholine or dimyristoylphosphatidylglycerol. It is concluded that the separation of the helices 1 and 5 constitutes one of the key steps along the complex pathway for the formation of the final apolipoprotein lipid-bound state. It is inferred that the conformational flexibility of helices 1 and 5 is a key property of apoLp-III, allowing the exposure of hydrophobic protein regions and the interaction of the hydrophobic faces of the amphipathic alpha-helices with the lipoprotein lipid surface.