The “beta-clasp” model of apolipoprotein A-I — A lipid-free solution structure determined by electron paramagnetic resonance spectroscopy

Abstract

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free / lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I.

Specific cholesterol binding drives drastic structural alterations in Apolipoprotein A1

“…have been experimentally found to have higher propensity for lipid binding. 11,22,25,[37][38][39][40] Our simulations are in excellent match with all these previous results and shows new regions of lipid-binding initiation in concentration gradient manner.…”

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

“…DMEM, H6(AB), H7(AB), H8(BB), H9(B), and H10(AB) (10). Sequence analysis (9,10), NMR assignments (11), hydrogen-deuterium exchange measurements (12,13), and site-directed spin-label electron paramagnetic resonance spectroscopy (14)(15)(16)(17) have provided different distributions, flexible regions, and positions for these putative helical tandem repeats in lipid-free or lipid-bound state. In addition, segment deletion and point mutation studies have further elucidated the possible conformation and function for each helical segment (18)(19)(20)(21)(22)(23)(24)(25)(26)(27).…”

Localization of APOL1 Protein and mRNA in the Human Kidney

“…15 The APOL1 concentrations in media to which our cell lines were exposed were far lower than those previously reported by Duchateau in serum (5-10 mg/ml) 16 and estimated in our studies (15 mg/ml). Assuming that #5% of serum APOL1 exists in a lipid-free form, as is the case for APOA1, 17,18 we estimate that approximately 0.75 mg/ml APOL1 could potentially cross the glomerular filtration barrier. This concentration is within the range used in our in vitro study, where we observed significant uptake of APOL1 by podocytes (Figure 8, Supplemental Figures 3, 5-7), but not by endothelial and mesangial cells, and is consistent with the observed enrichment of podocyte APOL1 signal in kidney cryosections.…”