Mechanisms of phospholipid/cholesterol translocation by ABCA1

摘要（项目3）

SUMMARY (Project 3) The ATP-binding cassette transporter A1 (ABCA1) is critical for the production of high-density lipoproteins (HDL) and, in turn, for all the protective functions of that lipoprotein family. Numerous attempts have been made to boost ABCA1-mediated lipid transport activity through various cellular and extracellular mechanisms. For a variety of reasons, few of these have made it into the clinic. We believe that a major factor underlying the failure to effectively exploit the ABCA1 pathway clinically is a fundamental lack of understanding of how the transporter works at the molecular level. Taking a mechanistic and structural approach, both in silico and in wet bench experiments, the main goal of Project 3 is to delineate the molecular mechanism behind ABCA1’s action. Our work in the previous cycle of this Program Project led to a 3-step model that serves as the central hypothesis of this work. Step 1: ABCA1 extracts lipid from the host cell’s outer membrane leaflet and moves it into a hydrophobic tunnel in its extracellular domain (Segrest et al., ABCA1 is an extracellular PL translocase, Na- ture Communications, 2022). Step 2: At a critical concentration of translocated lipid, ABCA1 dimerizes to com- plete the formation of a lipid receptacle that fills with lipid. Step 3: The extracellular lipid receptacle creates a highly specific dimeric binding site for APOA1 so that lipid extruded from the receptacle creates nascent HDL. Our model is supported by MD studies that support the targeting of extracellular leaflet lipid and its accumulation in a remote receptacle, by our refinement of the cryo-EM structure, which now strongly supports lipid extraction from the outer leaflet, and by mutagenesis of key residues in ABCA1 and APOA1 driven by our models. Importantly, our model explains observations that have been used to support both the direct binding and the microsolubiliza- tion mechanisms. Aim 1 will integrate in silico and in vitro approaches to test four key aspects of our model. i) We will experimentally determine if ABCA1 extracts lipids from the extracellular leaflet of the cell membrane and will use MD studies to elucidate whether natural sequence variants in ABCA1 support the outer leaflet lipid extraction model. ii) We will use MD simulations to visualize the effects of adding PLs into ABCA1’s proposed lipid recepta- cle domain and evaluate the receptacle as a potential binding site for amphipathic helical apolipoproteins. iii) We will test the requirement for ABCA1 dimerization via a series of point mutations predicted to alter dimerization. iv) We will model APOA1’s interactions with a ‘clasp’ domain of ABCA1 that we believe is critical for HDL assembly. Aim 2 presents direct evidence for ABCA1 self-interaction that is stable to detergent from cryo-EM data and pro- poses to reconstruct the dimer in bilayers. We will reconstitute self-interacting ABCA1 into unilammelar liposomes and novel oversized nanodisc platforms and characterize by cryo-EM. We will exploit our ABCA1 reconstitution system to perform isotope-assisted chemical crosslinking experiments to: i) test intramolecular contacts predicted, ii) verify that ABCA1 dimerizes, and test predicted intermolecular contacts, and iii) identify sites on both lipid-free and lipid-associated APOA1 and ABCA1 that mediate interactions between the two molecules.