A molecular trigger of lipid binding-induced opening of a helix bundle exchangeable apolipoprotein

Apolipophorin III (apoLp-III) from the sphinx moth, Manduca sexta, is a hel ix bundle protein that interacts reversibly with lipoproteins. Its five elo ngated amphipathic cy-helices are organized in an antiparallel fashion, wit h helices 3 and 4 connected by a short 6-residue (PDVEKE) linker helix, ter med helix 3'. Upon interaction with lipoproteins, apoLp-III opens to expose a continuous hydrophobic interior, It was postulated that helix bundle ope ning is preceded by an initiation step wherein helix 3' serves to recognize available lipoprotein surface binding sites. To test this hypothesis, heli x 3' was replaced by residues that have a propensity to form a type I beta- turn, NPNG, This mutant apoLp-III. was defective in lipoprotein binding ass ays. To define a more precise mode of interaction, the relevance of the pre sence of the hydrophobic Val-97 flanked by Asp-96 and Glu-98 was investigat ed by site-directed mutagenesis. V97N and D96N/V97N/E98Q apoLp-III were una ble to compete with wild-type apoLp-III to initiate an interaction with lip oproteins, whereas D96N/E98Q apoLp-III was as competent as wild-type apoLp- III. The results suggest that Val-97 is critical, whereas Asp-96 and GLu-98 are irrelevant for initiating binding to lipoproteins. A model of binding is presented wherein apoLp-III is oriented with the helix 3' end of the mol ecule juxtaposed to the lipoprotein surface. Recognition of lipoprotein sur face hydrophobic defects by Val-97 triggers opening of the helix bundle and facilitates formation of a stable binding interaction.