Mechanisms of phospholipid/cholesterol translocation by ABCA1

摘要(项目3) 三磷酸腺苷结合盒转运体A1(ABCA1)对高密度脂蛋白的产生至关重要 (高密度脂蛋白)，进而对该脂蛋白家族的所有保护功能起作用。已经进行了无数次尝试 通过各种细胞和细胞外机制增强ABCA1介导的脂质转运活性。 由于各种原因，很少有这种药物进入临床。我们认为，一个主要因素是 未能在临床上有效地开发ABCA1途径是对如何在临床上 转运蛋白在分子水平上起作用。采用机械和结构方法，在硅胶和湿法中都是如此 台架实验，项目3的主要目标是描绘ABCA1的S背后的分子机制 行动。我们在此计划项目的前一个周期中所做的工作导致了作为中心的3步模型 这项工作的假设。第一步：ABCA1从宿主细胞的外膜叶中提取脂类并移动它 进入其胞外区的疏水隧道(Segrest等人，ABCA1是一种胞外PL转位酶，Na- True Communications，2022)。第二步：在转位脂质的临界浓度下，ABCA1二聚为COM- 形成充满脂类的脂类容器。第三步：细胞外脂容器产生一种 APOA1的高度专一性二聚体结合部位，使脂类从容器中挤出，产生新生的高密度脂蛋白。我们的 模型得到了MD研究的支持，这些研究支持细胞外小叶脂的靶向及其在 远程容器，通过我们对冷冻-EM结构的改进，它现在强烈支持从 外层小叶，以及由我们的模型驱动的ABCA1和APOA1关键残基的突变。重要的是 我们的模型解释了被用来支持直接结合和微溶解的观察结果。 作用机制。目标1将集成在计算机和体外方法中，以测试我们模型的四个关键方面。I)我们 将从实验上确定ABCA1是否从细胞膜的胞外小叶中提取脂类，并将 使用MD研究来阐明ABCA1的自然序列变异是否支持外叶脂肪的提取 模特。Ii)我们将使用MD模拟来可视化向ABCA1的S提出的脂质接受器中添加PLS的效果- CLE结构域，并评估该受体作为两亲性螺旋载脂蛋白的潜在结合部位。三)我们 将通过一系列预计会改变二聚化的点突变来测试ABCA1二聚化的要求。四) 我们将对APOA1‘S与ABCA1的一个’CLAP‘结构域的相互作用进行建模，我们认为该结构域对高密度脂蛋白的组装至关重要。 从冷冻-EM数据和PRO-EM数据中，AIM 2提供了ABCA1自我相互作用的直接证据，该相互作用对洗涤剂稳定 在双层中重建二聚体的姿势。我们将把自我相互作用的ABCA1重组成单胺类脂质体 和新颖的超大尺寸纳米盘平台，并通过冷冻-EM进行表征。我们将利用我们的ABCA1重组 用于执行同位素辅助化学交联实验以：i)测试预测的分子内接触的系统， Ii)验证ABCA1二聚化，并测试预测的分子间接触，以及iii)确定两个无脂分子上的位置 以及脂质相关的APOA1和ABCA1，它们介导这两个分子之间的相互作用。

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.