Structure-function relationships in apolipoprotein A-I: Functional importance of the helix registry in discoidal HDL

PRIETO ED; CUELLAR LA; CABALEIRO LV; GARDA HA

La Plata

Encuentro; Humboldt Kollege 2010. Argentine-Germany: A century of scientific cooperation in Physics; 2011

Alexander von Humboldt Foundation

Apolipoprotein A-I (apoAI), the main protein of high density lipoproteins (HDL), plays a key role in cholesterol homeostasis and prevention of cardiovascular diseases. This amphitropic protein exchanges among a lipid-free and different lipid-bound forms during its functional cycle. It is composed of a series of amphipathic a-helical repeats that fold in a globular bundle. Upon lipid binding, the bundle is opened and helix-helix interactions are substituted by helix-lipid interactions. Most of the amphipathic a-helical repeats have a type A charge distribution in the helix polar face. This charge distribution allows the interaction with membrane or HDL surfaces with the long helix axis parallel to the lipid surface, forming electrostatic interactions with the phospholipid polar groups and hydrophobic interactions with the hydrocarbon chains. However, some a-helical repeats as the 3-4 pair, have a different charge distribution at their polar face and behave differently. We have previously detected (J Biol Chem 276:16978, 2001) that this apoAI region inserts very deeply into the membrane lipid bilayer. More recently, we determined the membrane insertion topology and proposed that an intermolecular bundle of both 3-4 helix pairs in a membrane bound apoAI dimer is inserted with the long helix axis roughly perpendicular to the membrane surface (Biochemistry 50:466, 2011). Discoidal HDL complexes (dHDL) are key intermediates in HDL biogenesis. The proposed intermolecular bundle of the 3-4 helix pairs can also be formed in dHDL, where it could act as a reversible membrane-anchoring domain to modulate lipid exchange with membranes. Most of the present knowledge of dHDL is based on studies with reconstituted complexes of dimeric apoAI obtained by cholate-dialysis. However, dHDL can also be generated by the spontaneous reaction with phospholipid vesicles at the temperature range of the gel/liquid-crystalline phase transition, a process that could be mechanistically similar to the "in vivo" apoAI lipidation. Recent studies from our lab evidenced that spontaneously generated dHDL are more active in promoting cell cholesterol efflux than those obtained by cholate dialysis. Measurements of intermolecular distances by fluorescence resonance energy transfer (FRET) in dimeric dHDL reconstituted with a series of single tryptophan mutants and extrinsically labeled cysteine mutants, indicate that: a) Dimeric dHDL obtained by cholate dialysis are a mixture of coexisting configurations with variable helix registry. As reported by others, they contain a main population with helices 5 of each monomer in juxtaposition (LL5/5 configuration) and a minor population with helix 5 of one monomer in contact with helix 2 of the other (LL5/2 registry); and b) In dimeric dHDL spontaneously generated at the phospholipid phase transition, data indicate the existence of only one population with the LL5/2 configuration. Together, these data indicate that only the LL5/2 configuration is active in promoting cell cholesterol efflux. This fact can be explained on the basis of the intermolecular bundle of the 3-4 helix pairs that can only be formed with the LL5/2, but not with the LL5/5 registry.