Mechanisms of phospholipid/cholesterol translocation by ABCA1

摘要（项目3） ATP结合盒转运蛋白A1（ABCA 1）是高密度脂蛋白产生的关键 (HDL)反过来，也会影响脂蛋白家族的所有保护功能。已经有许多尝试 通过各种细胞和细胞外机制增强ABCA 1介导的脂质转运活性。 由于各种原因，其中很少有人进入诊所。我们认为， 未能在临床上有效地利用ABCA 1通路是一个根本性的缺乏了解如何 在分子水平上起作用。采用机械和结构方法，无论是在电脑上还是在湿 实验室实验，项目3的主要目标是描绘ABCA 1的分子机制背后的 行动上我们在本计划项目的上一个周期中的工作导致了一个三步模型，该模型作为核心 这项工作的假设。步骤1：ABCA 1从宿主细胞的外膜小叶中提取脂质并将其移动 进入其细胞外结构域中的疏水通道（Segrest等，ABCA 1是一种细胞外PL移位酶， True Communications，2022）。步骤2：在临界浓度的易位脂质，ABCA 1二聚化，以com-bind。 形成充满脂质的脂质容器。步骤3：细胞外脂质容器产生一个 高特异性的二聚体结合位点的载脂蛋白A1，使脂质挤出容器产生新生的HDL。我们 该模型得到了MD研究的支持，这些研究支持靶向细胞外小叶脂质及其在细胞内的蓄积。 远程容器，通过我们对冷冻EM结构的改进，现在强烈支持从 外叶，并通过我们的模型驱动的ABCA 1和APOA 1的关键残基的诱变。重要的是， 我们的模型解释了用于支持直接结合和微溶解的观察结果， 作用机制。目标1将整合在硅片和体外方法来测试我们的模型的四个关键方面。i）我们 将通过实验确定ABCA 1是否从细胞膜的细胞外小叶提取脂质，并将 使用MD研究来阐明ABCA 1中的天然序列变体是否支持外叶脂质提取 模型ii）我们将使用MD模拟来可视化将PL添加到ABCA 1提出的脂质受体中的效果。 cle结构域和评估作为一个潜在的结合位点的两亲性螺旋载脂蛋白的插座。（三）我们 将测试通过一系列预测改变二聚化的点突变对ABCA 1二聚化的需求。iv）第四条 我们将模拟APOA 1与ABCA 1的“扣”结构域的相互作用，我们认为这对HDL组装至关重要。 目的2提供了ABCA 1自身相互作用的直接证据，该相互作用对来自冷冻EM数据的洗涤剂稳定，并且对来自冷冻EM数据的洗涤剂稳定。 构成重建双层中的二聚体。我们将自身相互作用的ABCA 1重组到单层脂质体中 和新的超大纳米盘平台，并通过冷冻-EM表征。我们将利用我们的ABCA 1重组 进行同位素辅助化学交联实验的系统，以：i）测试预测的分子内接触， ii）验证ABCA 1二聚化，并测试预测的分子间接触，和iii）鉴定两种无脂质 以及介导两种分子之间相互作用的脂质相关的APOA 1和ABCA 1。

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.