α-helix formation is required for high affinity binding of human apolipoprotein A-I to lipids

Abstract

Apolipoprotein (apo) A-I is thought to undergo a conformational change during lipid association that results in the transition of random coil to α-helix. Using a series of deletion mutants lacking different regions along the molecule, we examined the contribution of α-helix formation in apoA-I to the binding to egg phosphatidylcholine (PC) small unilamellar vesicles (SUV). Binding isotherms determined by gel filtration showed that apoA-I binds to SUV with high affinity and deletions in the C-terminal region markedly decrease the affinity. Circular dichroism measurements demonstrated that binding to SUV led to an increase in α-helix content, but the helix content was somewhat less than in reconstituted discoidal PC-apoA-I complexes for all apoA-I variants, suggesting that the helical structure of apoA-I on SUV is different from that in discs. Isothermal titration calorimetry showed that the binding of apoA-I to SUV is accompanied by a large exothermic heat and deletions in the C-terminal regions greatly decrease the heat. Analysis of the rate of release of heat on binding, as well as the kinetics of quenching of tryptophan fluorescence by brominated PC, indicated that the opening of the N-terminal helix bundle is a rate-limiting step in apoA-I binding to the SUV surface. Significantly, the correlation of thermodynamic parameters of binding with the increase in the number of helical residues revealed that the contribution of α-helix formation upon lipid binding to the enthalpy and the free energy of the binding of apoA-I is -1.1 and -0.04 kcal/mol per residue, respectively. These results indicate that α-helix formation, especially in the C-terminal regions, provides the energetic source for high affinity binding of apoA-I to lipids.