Structural basis for the conformational adaptability of apolipophorin III, a helix-bundle exchangeable apolipoprotein

Abstract

The high-resolution NMR structure of apolipophorin III from the sphinx moth, Manduca sexta, has been determined in the lipid-free state. We show that lipid-free apolipophorin III adopts a unique helix-bundle topology that has several characteristic structural features. These include a marginally stable, up-and-down helix bundle that allows for concerted opening of the bundle about "hinged" loops upon lipid interaction and buried polar/ionizable residues and buried interhelical H-bonds located in the otherwise hydrophobic interior of the bundle that adjust protein stability and facilitate lipid-induced conformational opening. We suggest that these structural features modulate the conformational adaptability of the lipid-free helix bundle upon lipid binding and control return of the open conformation to the original lipid-free helix-bundle state. Taken together, these data provide a structural rationale for the ability of exchangeable apolipoproteins to reversibly interact with circulating lipoprotein particles.

Figure 1

NMR structure of lipid-free M. sexta apoLp-III. (A and B) Superposition of 40 best-fit NMR-derived structures of apoLp-III, with backbone atoms displayed in white and side-chain heavy atoms displayed in green. (C) Ribbon representation of an energy minimized, average structure of apoLp-III (PDB code 1EQ1). A and B were prepared by using insightii (Molecular Simulations, Waltham, MA), and C was prepared by using molmol (23).

Figure 2

Helix-bundle topology and conformational opening. In the apoLp-III helix bundle (B), short loops connecting long helices serve as hinges, about which helical segments in the bundle reposition, adopting an extended open α-helical structure. By contrast, in cytochrome c (A, PDB code 1CCH), the up-and-down helix bundle is connected by a long loop with a short β-strand, and in IL-4 (C, PDB code 1RCB), the up-up-down-down helix bundle is connected by long loops and a short β-strand. Opening of these helix bundles is hindered by the molecular organization of their respective bundles.

Figure 3

Helix wheel diagram of the apoLp-III five-helix bundle. Each circle represents one residue and is labeled by sequence number and amino acid. The color code indicates residues with different ASA values based on vadar calculation. Residues have been classified as indicated.

Figure 4

Interhelical H-bonds in the helix bundle. Top view (Upper) and bottom view (Lower) of the helix bundle. Buried H-bonds are highlighted by pink dotted lines. Light blue helices are helices 3 and 4, green helices are helices 1, 2, and 5.