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J Biol Chem, Vol. 274, Issue 31, 21804-21810, July 30, 1999

Interaction of an Exchangeable Apolipoprotein with Phospholipid Vesicles and Lipoprotein Particles
ROLE OF LEUCINES 32, 34, AND 95 IN LOCUSTA MIGRATORIA APOLIPOPHORIN III

Paul M. M. Weers, Vasanthy Narayanaswami, Cyril M. Kay^¶, and Robert O. Ryan

From the  Lipid and Lipoprotein Research Group, ^¶ Protein Engineering Network of Excellence, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipid surfaces. In the lipid-free state this 164-residue protein exists as a bundle of five elongated amphipathic -helices. Upon lipid binding, apoLp-III undergoes a significant conformational 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 proposed 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 initiation of lipid binding. To examine this hypothesis, mutant apoLp-IIIs were designed wherein the three Leu residues were replaced by Arg, individually or together. Circular dichroism spectroscopy and temperature and guanidine hydrochloride denaturation studies showed that the mutations did not cause major changes in secondary structure content or stability. In lipid binding assays, addition of apoLp-III to phospholipid vesicles caused a rapid clearance of vesicle turbidity due to transformation to discoidal complexes. L34R and L32R/L34R/L95R apoLp-IIIs displayed a much stronger interaction with lipid vesicles than wild-type apoLp-III. Furthermore, it was demonstrated 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 compared with wild-type apoLp-III. The data indicate that the loops connecting helices 1 and 2 and helices 3 and 4 are critical regions in the protein, contributing to recognition of hydrophobic defects on lipoprotein surfaces by apoLp-III.

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