Dynamics of LCAT activation and lipoprotein remodeling

描述（申请人提供）：高密度脂蛋白（HDL）是心血管疾病（CVD）药物治疗的有希望的靶点。无论HDL本身是直接预防心血管疾病还是作为抗炎或抗氧化蛋白附着的平台，了解HDL的结构都很重要。目前的目标是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HDL的两个重要生物学功能中的作用：1)卵磷脂酶的激活：胆固醇酰基转移酶（LCAT），在HDL组装过程中负责将新生（盘状）HDL转化为循环（球形）HDL的酶，这是逆向胆固醇转运（RCT）和ii) HDL重塑的重要步骤，在HDL组装和RCT中也很重要。由于apoA- I/HDL是一种软形式的凝聚态物质，容易因热波动而变形，因此更全面地了解HDL将需要创新的方法。原则上，我们建议使用实验方法和计算机模拟的协同组合可以显著有助于理解HDL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，我们提出了三个可行的假设：1)螺旋内和螺旋间盐桥的随机云分别为高密度脂蛋白颗粒上的apoA-I提供了类似弹簧的弹性（分子“Slinky”）和粘性（分子“Velcro”）。2) apoA-I在HDL上的末端结构域是调节极性脂质交换的重塑开关，并与其他载脂蛋白、抗炎和抗氧化蛋白形成高亲和力的热点。3) apoA-I的成对反平行螺旋5结构域为未酯化胆固醇的疏水酰基链和极性羟基从新生HDL迁移到LCAT的活性位点创造了一个两向递呈通道。为了验证这些假设，我们提出了两个具体目标：1)确定apoA-I末端重叠结构域在新生HDL重塑中的作用。为了实现这一目标，我们将：i)利用我们的MD结果通过位点定向诱变设计融合、交换、膜相互作用和蛋白质结合亲和力的分子模型的实验测试，重点关注N端“粘性”推定融合结构域和c端“混杂”螺旋10推定交换结构域。ii)使用apoA-I/HDL集合的所有原子和粗粒度模型（原生和突变），通过MD模拟进一步验证极性脂质重塑开关假说。2)检测apoA-I中心结构域在LCAT激活中的作用。酰基链和UC呈现通道假说将通过基于所有原子和粗粒度MD模拟设计的位点定向突变体进行实验验证。由于对脂质相关apoA-I结构的详细预测，我们提出的湿实验室方法与分子模拟的结合，以及我们独特的定位，可以为未来研究HDL结构-功能和动力学的分子机制提供分子路线图。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: HDL, the good cholesterol, is an important target for future drugs to prevent heart attacks. Unfortunately, all recent attempts at new HDL-targeted drug development have been unsuccessful. The combination of computer and molecular biology studies of HDL that we propose, a combination unique to our laboratory, provides a molecular blueprint for future drug development aimed at HDL.