Computational analysis of phosphoinositide signaling

描述(由申请人提供)：我们的长期目标是继续开发计算方法，在分子水平上定量描述导致膜表面形成大分子蛋白质/脂复合体的物理作用力。这些复合体在多种细胞过程中发挥作用，如界面信号转导。我们对当前提议的总体假设是，静电学在肌醇磷脂信号转导中发挥普遍作用，并通过肌醇磷脂介导的膜结合和蛋白质和脂类的侧向组织来表现。第一个具体目标是预测肌醇磷脂结合域的膜相关取向，并定量描述结合配体对这些结合域的静电性质的影响。描述磷脂酰肌醇如何改变这些结构域的静电特性对于理解膜结合是如何调控至关重要的：我们假设，高度负电荷的磷脂酰肌醇的结合中和了配体结合口袋中的碱性基团，降低了疏水基序插入膜界面的能量障碍。第二个具体目的是预测磷脂酰肌醇如何影响C2结构域的膜相关取向，从而促进钙介导的细胞过程，如突触囊泡胞吐。我们假设这些相互作用主要是由非特定静电力驱动的。第三个具体目标是模拟膜表面蛋白质和磷脂酰肌醇之间的横向相互作用，以表征在其组织上形成的多聚体结构。这类复合体在细胞内钙释放和细胞趋化中起关键作用。我们假设膜表面的局部正负电势区域是吸引具有相反静电特性的分子的盆地，静电是形成这些络合物的主要驱动力。我们的计算将为磷脂酰肌醇结合结构域的膜结合、膜介导的相互作用以及两者的调节提供可实验检验的假说。对静电相互作用的全面描述将为将来研究其他重要的相互作用铺平道路。影响膜靶向的突变与癌症和微生物易感性有关。因此，这项研究将提供对正常和异常功能的洞察。

DESCRIPTION (provided by applicant): Our long-term goal is to continue the development of computational methods to describe quantitatively, at the molecular level, the physical forces responsible for the formation of macromolecular protein/lipid complexes at membrane surfaces. These complexes function in a variety of cellular processes such as interfacial signal transduction. Our overall hypothesis for the current proposal is that electrostatics plays a general role in phosphoinositide signaling and is manifested through phosphoinositide-mediated membrane association and the lateral organization of proteins and lipids. The first Specific Aim is to predict the membrane-associated orientations of phosphoinositide-binding domains and to describe quantitatively the effect of bound ligand on the electrostatic properties of these domains. Describing how phosphoinositides change the electrostatic character of these domains is crucial to understanding how membrane association is regulated: We hypothesize that binding of the highly negatively charged phosphoinositide neutralizes basic groups in the ligand-binding pocket and reduces the energetic barrier to insertion of hydrophobic motifs into the membrane interface. The second Specific Aim is to predict how phosphoinositides affect the membrane- associated orientations of C2 domains and, thus, contribute to calcium-mediated cellular processes such as synaptic vesicle exocytosis. We hypothesize that these interactions are driven mainly by non-specific electrostatic forces. The third Specific Aim is to simulate the lateral interactions among proteins and phosphoinositides at membrane surfaces in order to characterize the multimeric structures that form upon their organization. Such complexes play key roles in intracellular calcium release and cellular chemotaxis. We hypothesize that localized regions of negative and positive potential at the membrane surface serve as basins of attraction for molecules with opposite electrostatic characteristics and that electrostatics is a major driving force in forming these complexes. Our calculations will provide experimentally testable hypotheses for the membrane association of phosphoinositide binding domains, for membrane-mediated interactions, and for the regulation of both. A full description of electrostatic interactions will pave the way for the future study of other important interactions. Mutations that affect membrane targeting are implicated in cancer and microbial susceptibility. Thus, this research will provide insight into both normal and aberrant functionality.