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

Abstract

Apolipophorin III (apoLp-III) from the sphinx moth, Manduca sexta, is a helix bundle protein that interacts reversibly with lipoproteins. Its five elongated amphipathic alpha-helices are organized in an antiparallel fashion, with helices 3 and 4 connected by a short 6-residue (PDVEKE) linker helix, termed helix 3'. Upon interaction with lipoproteins, apoLp-III opens to expose a continuous hydrophobic interior. It was postulated that helix bundle opening is preceded by an initiation step wherein helix 3' serves to recognize available lipoprotein surface binding sites. To test this hypothesis, helix 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 assays. To define a more precise mode of interaction, the relevance of the presence of the hydrophobic Val-97 flanked by Asp-96 and Glu-98 was investigated by site-directed mutagenesis. V97N and D96N/V97N/E98Q apoLp-III were unable to compete with wild-type apoLp-III to initiate an interaction with lipoproteins, 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 molecule juxtaposed to the lipoprotein surface. Recognition of lipoprotein surface hydrophobic defects by Val-97 triggers opening of the helix bundle and facilitates formation of a stable binding interaction.

Figure 1

Schematic representation of M. sexta apoLp-III. The tertiary fold of apoLp-III reveals a five-helix bundle, where the hydrophobic faces of the amphipathic helices are oriented inwards. Helix 3′ (95PDVEKE100) connects helices 3 and 4 and lies outside the context of the bundle.

Figure 2

Competition between WT and Δ93–102/NPNG apoLp-III for binding to PL-C-treated LDL. LDL (250 μg of protein) was incubated with 880 mU of PL-C in the presence of WT (250 μg), Δ93–102/NPNG (250 μg), or both WT and Δ93–102/NPNG apoLp-III (250 μg of each) at 37°C. After 40 min, unbound apoLp-III was separated from LDL-bound apoLp-III. The LDL-containing (lanes 1, 2, and 3), and the bottom fractions (lanes 6, 7, and 8) were analyzed by SDS/PAGE. The lanes contained PL-C-treated LDL plus the following: WT apoLp-III (lanes 1 and 6); Δ93–102/NPNG apoLp-III (lanes 2 and 7); and WT and Δ93–102/NPNG apoLp-III (lanes 3 and 8). Standard WT and Δ93–102/NPNG apoLp-III were in lanes 4 and 5, respectively.

Figure 3

Competition between WT apoLp-III and point mutants for initiation of lipoprotein binding. LDL (250 μg of protein) was incubated at 37°C with ³H-labeled WT apoLp-III (250 μg of culture medium protein) and 880 mU of PL-C in the absence (bar a) or presence of unlabeled competitor apoLp-III: WT (bar b), Δ93–102/NPNG (bar c), V97N (bar d), D96N/E98Q (bar e), or D96N/V97N/E98Q (bar f) (250 μg each). After 40 min of incubation, unbound apoLp-III was separated from LDL-bound apoLp-III by density gradient ultracentrifugation, and the radioactivity in the bound fraction was determined. The bars represent percentage of LDL-bound ³H-labeled WT apoLp-III in the presence of competitor compared with that in the absence of competitor apolipoprotein (taken as 100%). A control incubation, with no added PL-C, was included to estimate nonspecific binding of ³H-labeled WT apoLp-III (<3%; not shown).

Figure 4

Binding and stabilization of PL-C-treated LDL by apoLp-III. (A) LDL (50 μg of protein) was incubated at 37°C with 160 mU of PL-C in the absence (curve a) or presence (50 μg each) of apolipoproteins, WT (curve b), Δ93–102/NPNG (curve c), V97N (curve d), D96N/V97N/E98Q (curve e), or D96N/E98Q (curve f). Control incubation containing no PL-C (curve g) was included. Sample absorbance was measured at 340 nm. (B) Incubations were the same as for A, except incubations for curves b and d, in which KBr (0.25 M, final concentration) was added after 30 min (see arrow), to inhibit PL-C activity (19). LDL was treated with PL-C in the absence (curves a and b) or the presence of V97N apoLp-III (curves c and d).

Figure 5

Model of apoLp-III binding interaction (not to scale). In the lipid-free state, apoLp-III exists as a closed five-helix bundle, poised to bind to lipophorin. Appearance of DAG in the lipoprotein surface monolayer induces apoLp-III binding at its helix 3′ end, which triggers helix-bundle opening. This opening ultimately leads to replacement of helix–helix contacts in the bundle conformation by helix–lipid contacts in the lipoprotein-bound state.