Insight into lipid surface recognition and reversible conformational adaptations of an exchangeable apolipoprotein by...

Apolipophorin III (apoLp-III) from the insect Manduca sexta is a 166-residue (Mr 18,340) member of the exchangeable apolipoprotein class that functions to stabilize lipid-enriched plasma lipoproteins. In the present study, we present the secondary structure and global fold of recombinant apoLp-III derived from three-dimensional heteronuclear NMR spectroscopy experiments. Five discrete alpha-helical segments (21-30 residues in length) with well defined boundaries were characterized by four NMR parameters: medium range nuclear Overhauser enhancement contacts between proton pairs, chemical shift index, coupling constants, and amide proton exchange rates. An antiparallel arrangement of helical segments has been obtained based on the long range interhelical nuclear Overhauser enhancement contacts. The NMR solution structure reveals a globular, up and down helix bundle organization similar to that of Locusta migratoria apoLp-III (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). However, a short helix (comprised of 5 amino acids) has been identified in the region between helix 3 and helix 4. This helix is postulated to play a role in lipid surface recognition and/or initiation of binding. Our results also indicate the existence of buried polar and charged residues in the helix bundle, providing a structural basis for the relatively low stability of apoLp-III in its lipid-free state. It is suggested that the intrinsic low stability of lipid-free apoLp-III may be important in terms of its ability to undergo a reversible, lipid binding-induced, conformational change. This study underscores the striking resemblance in molecular architecture between insect apoLp-III and the N-terminal domain of human apolipoprotein E. The potential for application of NMR techniques to studies of the exchangeable apolipoproteins, possibly in their biologically active, lipid-associated state, has broad implications in terms of our understanding of the molecular basis of their physiological functions.