Interaction of an exchangeable apolipoprotein with phospholipid vesicles and lipoprotein particles - Role of leucines 32, 34, and 95 in Locusta migratoria apolipophorin III

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable ap olipoprotein that binds reversibly to lipid surfaces. In the lipid-free sta te this 164-residue protein exists as a bundle of five elongated amphipathi c alpha-helices, Upon lipid binding, apoLp-III undergoes a significant conf ormational change, resulting in exposure of its hydrophobic interior to the lipid environment. On the basis of x-ray crystallographic data (Breiter, D . R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A , Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608), it was p roposed that hydrophobic residues, present in loops that connect helices 1 and 2 (Leu-32 and Leu-34) and helices 3 and 4 (Leu-95), may function in ini tiation of lipid binding. To examine this hypothesis, mutant apoLp-IIIs wer e designed wherein the three Leu residues were replaced by Arg, individuall y or together. Circular dichroism spectroscopy and temperature and guanidin e hydrochloride denaturation studies showed that the mutations did not caus e major changes in secondary structure content or stability. In lipid bindi ng assays, addition of apoLp-III to phospholipid vesicles caused a rapid cl earance of vesicle turbidity due to transformation to discoidal complexes. L34R and L32R/L34R/L95R apoLp-IIIs displayed a much stronger interaction wi th lipid vesicles than wild-type apoLp-III. Furthermore, it was demonstrate d that the mutant apoLp-IIIs retained their ability to bind to lipoprotein particles. However, in lipoprotein competition binding assays, the mutants displayed an impaired ability to initiate a binding interaction when compar ed with wild-type apoLp-III, The data indicate that the loops connecting he lices 1 and 2 and helices 3 and 4 are critical regions in the protein, cont ributing to recognition of hydrophobic defects on lipoprotein surfaces by a poLp-III.