Apolipoprotein A-I helical structure and stability in discoidal high-density lipoprotein (HDL) particles by hydrogen exchange and mass spectrometry

Abstract

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6 nm) and small (7.8 nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5 kcal∕mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.atherosclerosis | amphipathic α-helix | protein secondary structure T here is great interest in understanding the structure-function relationships of high-density lipoprotein (HDL) because of its important antiatherogenic properties. Because high-resolution structures of HDL microemulsion particles cannot be obtained by current X-ray crystallography and NMR methods, alternative biophysical approaches have been used to characterize various subspecies of HDL. Structural models that show the general lipid and protein organization in HDL particles are now available (for reviews, see refs. 1-6). To derive detailed understanding at the molecular level of how HDL functions in cholesterol transport (7) and in reducing the incidence of premature cardiovascular disease (8), higher-resolution structural information is required.Reconstituted discoidal HDL particles that are models of nascent HDL (9) created by the interaction of apolipoprotein (apo) A-I (3, 10), the principal protein of HDL, with the cellsurface ATP binding cassette transporter (ABCA1) (11, 12) have received a great deal of attention. A major advantage of model particles is the possibility of obtaining preparations that are sufficiently homogeneous for detailed structural investigation. The structure of a discoidal HDL particle (approximately 10 nm hydrodynamic diameter) comprising a 16...

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

“…Indeed, this registry for the two antiparallel apoA1 chains is needed to accommodate the is an unstructured loop and not a helical domain as was inferred indirectly by Sevugan Chetty et al ( 24 ) from their HDX data (5°C) and the overall predicted ␣ -helical content of apoA1 based on circular dichroism data on nHDL POPC .…”

“…Before studies employing contrast variation small-angle neutron scattering (SANS) (see below), the overall conformation of apoA1 within a lipid containing HDL particles was inferred from crystal structure data from lipid free apoA1 mutant forms ( 19,20 ), as well as measured distance constraints between inter-and intrachain amino acids in mutated or derivatized apoA1 using FRET or ESR ( 12 ), and in native apoA1 by cross-linking MS studies ( 11,15,21,22 ). Further structural information has been obtained from amide bond hydrogen/deuterium exchange mass spectrometry ( 14,23,24 )}, and cryo-electron tomography ( 25 ).…”

ApoA1 and ApoA1-specific self-antibodies in cardiovascular disease

“…In HDL disks, amphipathic α-helices of ApoA1 are relatively unstable and can dynamically change the conformation from folded to unfolded 47 due to the presence of phospholipids. Indeed, the conformation of lipid-bound ApoA1 is highly flexible.…”

“…In contrast, a.a. 159-180 that are involved in the lecithincholesterol acyltransferase (LCAT)-activating region of ApoA1 are organized rather as a helix, but not as a random loop. 47 In smaller particles (7.8 nm), due to increased packing density of ApoA1, more regions (a.a. 45-57, 115-158, and 190-243) lose the helical structure and become unfolded. These regions correspond to the portions of lipid-free ApoA1 that are mostly non-helical.…”

“…These regions correspond to the portions of lipid-free ApoA1 that are mostly non-helical. 47 Discoidal HDL particles with the size exceeding 10 nm may include more ApoA1 molecules. In particles with three molecules, at least one should have a helical hairpin conformation.…”