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良好靶点。 (CVD)。高密度脂蛋白本身是直接预防心血管疾病还是作为附着保护性物质的平台 抗炎或抗氧化蛋白质，高密度脂蛋白结构的知识是重要的。当前的目标是 建议使用直接实验方法和计算机模拟的协同组合来 了解载脂蛋白A-I(apoA-I)动力学在高密度脂蛋白两个重要生物学功能中的作用 卵磷脂：胆固醇酰基转移酶(LCAT)的激活，该酶负责转化 在高密度脂蛋白组装过程中，新生的(盘状)高密度脂蛋白转变为循环的(球形)高密度脂蛋白，这是反向的重要一步 胆固醇转运(RCT)和II)高密度脂蛋白重塑，在高密度脂蛋白组装和高密度脂蛋白重塑中也很重要。因为阿波亚- I/hdl是一种软形式的凝聚态物质，容易受到热波动的变形，是一种更完整的 对高密度脂蛋白的理解将需要创新的方法。原则上，我们建议使用一种协同 实验方法和计算机模拟的结合可以大大有助于理解 高密度脂蛋白的结构和动力学。基于我们最近对高密度脂蛋白的分子动力学(MD)模拟，我们 提出三个工作假设：1)螺旋内和螺旋间盐桥的随机云， 分别为apoA-I提供弹力式弹性(分子“Slinky”)和粘性(分子“魔术贴”) 在高密度脂蛋白颗粒上。2)高密度脂蛋白上apoA-I的末端结构域代表一种重塑开关，它调节 交换极性脂类，并产生与其他载脂蛋白和抗炎蛋白高亲和力的热点 和抗氧化性蛋白质。3)apoA-I的成对反平行螺旋5结构域创建了一个两亲性 非酯化产物疏水酰链和极性羟基迁移的呈递通道 胆固醇从新生的高密度脂蛋白转移到LCAT的活性部位。为了检验这些假设，我们提出了两个具体的假设 目的：1)确定apoA-I末端重叠区在新生高密度脂蛋白重塑中的作用。至 为了实现这一目标，我们将：i)利用我们的MD结果设计实验测试，通过定点突变 融合、交换、膜相互作用和蛋白质结合亲和力的分子模型，侧重于N- 末端“粘性”的推定融合结构域和C-末端的“混杂”螺旋10推定的交换域。 二)使用apoA-I/高密度脂蛋白系综的所有原子和粗粒度模型(原生和突变)，以进一步测试 MD模拟的极脂重塑-开关假说。2)测试中央结构域的作用 载脂蛋白A-I在LCAT激活中的作用。将检验酰基链和UC呈现隧道假说 实验上，在所有原子和粗粒MD模拟的基础上设计了定点突变体。 由于对脂类相关载脂蛋白A-I结构的详细预测，湿实验室的组合方法 通过我们提出的和我们唯一定位的分子模拟可以提供一个分子 未来研究高密度脂蛋白结构-功能和动力学的分子机制的路线图。

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.