Dynamics of LCAT activation and lipoprotein remodeling

高密度脂蛋白（HDL）是心血管疾病药物治疗的重要靶点 （CVD）。HDL本身是否直接预防CVD或作为一个平台， 作为抗氧化蛋白，HDL结构的知识很重要。当前的目标 建议是使用直接实验方法和计算机模拟的协同组合， 了解载脂蛋白A-I（apoA-I）动力学在HDL的两个重要生物学功能中的作用：i） 激活酶卵磷脂：胆固醇酰基转移酶（LCAT），该酶负责转化 在HDL组装过程中，新生（盘状）HDL向循环（球状）HDL转化，这是逆转的一个重要步骤。 胆固醇转运（RCT）和ii）HDL重塑，在HDL组装和RCT中也很重要。因为载脂蛋白A- I/HDL是一种软态凝聚态物质，很容易因热涨落而变形， 对HDL的理解需要创新的方法。原则上，我们建议使用协同 实验方法和计算机模拟的结合可以大大有助于理解 HDL结构和动力学基于我们最近对HDL的分子动力学（MD）模拟，我们 提出了三个工作假设：1）螺旋内和螺旋间盐桥的随机云， 分别为apoA-I提供类似弹簧的弹性（分子“Slinky”）和粘性（分子“Velcro HDL颗粒。2)HDL上apoA-I的末端结构域代表了一个重塑开关， 极性脂质的交换，并产生对其他载脂蛋白和脂蛋白具有高亲和力的热点。 和抗氧化蛋白质。3)apoA-I的成对反平行螺旋5结构域产生两亲性的 用于未酯化的疏水酰基链和极性羟基迁移的呈现隧道 胆固醇从新生HDL转移到LCAT的活性位点。为了验证这些假设，我们提出了两个具体的假设。 目的：1）探讨apoA-I末端重叠区在新生HDL重构中的作用。到 为了实现这一目标，我们将：i）使用我们的MD结果设计实验测试，通过定点诱变， 融合，交换，膜相互作用和蛋白质结合亲和力的分子模型，重点是N- 末端“粘性”推定融合结构域和C末端“混杂”螺旋10推定交换结构域。 ii）使用apoA-I/HDL系综（天然的和突变的）的所有原子和粗粒度模型来进一步测试apoA-I/HDL系综的生物学特性。 极性脂质重塑开关假说的MD模拟。2)为了测试中心域的作用， apoA-I在LCAT激活中的作用。将检验酰基链和UC呈递隧道假设 实验上的定点突变体设计的基础上，所有原子和粗粒度的MD模拟。 由于脂质相关apoA-I结构的详细预测，湿实验室方法的组合 通过我们提出的分子模拟，我们处于独特的位置，可以提供一个分子 为HDL结构-功能和动力学的分子机制的未来研究路线图。

High density lipoproteins (HDL) are promising targets for pharmacological therapy of cardiovascular disease (CVD). Whether HDL itself directly prevents CVD or acts as a platform for attachment of protective antiinflammatory or antioxidant proteins, knowledge of HDL structure is important. The goal of the current proposal is to use a synergistic combination of direct experimental methods and computer simulations to understand the role of apolipoprotein A-I (apoA-I) dynamics in two important biological functions of HDL: i) activation of the enzyme lecithin:cholesterol acyl transferase (LCAT), the enzyme responsible for converting nascent (discoidal) HDL to circulating (spheroidal) HDL during HDL assembly, an important step in reverse cholesterol transport (RCT) and ii) HDL remodeling, also important in HDL assembly and RCT. Since apoA- I/HDL is a soft form of condensed matter easily deformable by thermal fluctuations, a more complete understanding of HDL will require innovative approaches. In principle, our proposed use of a synergistic combination of experimental methods and computer simulations can contribute significantly to understanding HDL structure and dynamics. Based upon our recent molecular dynamics (MD) simulations of HDL, we propose three working hypotheses: 1) A stochastic cloud of intrahelical and interhelical salt bridges, respectively, provide a spring-like elasticity (molecular "Slinky") and stickiness (molecular "Velcro") to apoA-I on HDL particles. 2) The terminal domains of apoA-I on HDL represent a remodeling-switch that regulates exchange of polar lipids and creates a hot spot with high affinity for other apolipoproteins and antiinflammatory and antioxidant proteins. 3) The pairwise antiparallel helix 5 domain of apoA-I creates an amphipathic presentation tunnel for migration of hydrophobic acyl chains and polar hydroxyl groups of unesterified cholesterol from nascent HDL to the active site of LCAT. To test these hypotheses we propose two specific aims: 1) To determine the role of the terminal overlap domain of apoA-I in nascent HDL remodeling. To achieve this aim, we will: i) Use our MD results to design experimental tests by site-directed mutagenesis of molecular models for fusion, exchange, membrane interactions and protein-binding affinity, focusing on the N- terminal "sticky" putative fusion domain and the C-terminal "promiscuous" helix 10 putative exchange domain. ii) Use all atom and coarse grained models of apoA-I/HDL ensembles (native and mutated) to further test the polar lipid remodeling-switch hypothesis by MD simulations. 2) To test the role of the central domain of apoA-I in LCAT activation. The acyl chain and UC presentation tunnel hypothesis will be tested experimentally by site-directed mutants designed on the basis of all atom and coarse grained MD simulations. Because of detailed predictions of lipid-associated apoA-I structure, the combination of wet lab approaches with molecular simulations that we propose and for which we are uniquely positioned can provide a molecular roadmap for future research into molecular mechanisms of HDL structure-function and dynamics.