Identification and Structural Ramifications of a Hinge Domain in Apolipoprotein A-I Discoidal High-density Lipoproteins of Different Size

Abstract

Apolipoprotein (apo) A-I is the major protein constituent of human high-density lipoprotein (HDL) and is likely responsible for many of its anti-atherogenic properties. Since distinct HDL size subspecies may play different roles in interactions critical for these properties, a key question concerns how apoA-I can adjust its conformation in response to changes in HDL particle size. A prominent hypothesis states that apoA-I contains a flexible "hinge domain" that can associate/dissociate from the lipoprotein as its diameter fluctuates. Although flexible domains clearly exist within HDL-bound apoA-I, this hypothesis has not been directly tested by assessing the ability of such domains to modulate their contacts with the lipid surface. In this work, discoidal HDL particles of different size were reconstituted with a series of human apoA-I mutants containing a single reporter tryptophan residue within each of its 22 amino acid amphipathic helical repeats. The particles also contained nitroxide spin labels, potent quenchers of tryptophan fluorescence, attached to the phospholipid acyl chains. We then measured the relative exposure of each tryptophan probe with increasing quencher concentrations. We found that, although there were modest structural changes across much of apoA-I, only helices 5, 6, and 7 exhibited significant differences in terms of exposure to lipid between large (96 A) and small (78 A) HDL particles. From these results, we present a model for a putative hinge domain in the context of recent "belt" and "hairpin" models of apoA-I structure in discoidal HDL particles.

Structure of HDL: Particle Subclasses and Molecular Components

“…The authors suggested that this region is responsible for absorbing changes in particle size by unfolding under conditions when lipid surface area is limiting. This is consistent with previous studies from our laboratory which used lipid-based fluorescence quenching agents to show that this same region changes its exposure to lipid when the particle diameter is reduced (Maiorano et al 2004).…”

The low-resolution structure of nHDL reconstituted with DMPC with and without cholesterol reveals a mechanism for particle expansion

“…The Belt model is characterized by a closed double belt conformation of the antiparallel apoA1 dimer encircling a lipid bilayer ( 9 ). Virtually all subsequent refi nements to models of nHDL (11)(12)(13)(14) have posited a closed apoA1 dimer in a belt confi guration that completely enshrines a central lipid bilayer, regardless of lipid composition. We recently used SANS with contrast variation to directly visu alize for the fi rst time the protein component of nHDL reconstituted with apoA1:POPC:cholesterol (1:100:10, Supplemental Material can be found at: micellar region).…”

“…Nevertheless, various models of nHDL have been proposed in the past ( 6,7 ), and they rely on biophysical, biochemical, and computer simulation studies [e.g., the Picket Fence model ( 8 ), the Belt model ( 9,10 ), the Hairpin Loop models (11)(12)(13), the Solar Flares model ( 14 ), the Double Super Helix model ( 15 ), and the Twisted Belt model ( 16,17 )]. All current models have in common an antiparallel orientation of the two apoA1 chains, but the overall conformation of apoA1 within the particle is still a matter of intense debate ( 18 ).…”

Apolipoprotein A-I Assumes a “Looped Belt” Conformation on Reconstituted High Density Lipoprotein

“…Recently, Maiorano and colleagues (7) proposed that apoA-I can adopt one of two possible extended ␣ helical conformations, including the extended belt model described above or a model wherein the apoA-I molecule loops back on itself, forming a "hairpin" structure ( Fig. 1).…”