Structure/Function of Terminal Complement Proteins

描述（由申请人提供）：本项目的目的是详细了解人C5 b-9的形成和功能，补体的溶细胞复合物称为“膜攻击复合物”或MAC。MAC是先天免疫系统的产物。MAC提供针对细菌感染的保护，但它也有助于炎性疾病的发病机制。MAC的组装通过补体组分C5 b、C6、C7、C8和C9之间的蛋白质-蛋白质和蛋白质-脂质相互作用的知之甚少的机制发生在靶细胞膜上。该项目将为这些相互作用的分子基础提供新的见解，并为设计MAC形成的抑制剂奠定基础。具体目标是： 1)确定人C8的晶体结构，这是一种151 kDa的蛋白质，由三个遗传上不同的亚基C81、C82和C83组成，它们排列为与C82非共价结合的二硫键连接的C81-3异二聚体。C81的中心40-kDa MACPF片段（1 MACPF）和与C83连接的1 MACPF（1 MACPF-3）的重组形式已经结晶，其结构部分解析。这些数据将有助于解决整个C8的晶体结构的X射线衍射数据（3.0 E分辨率）最近已经获得。 2)鉴定和表征C8上功能重要的结合位点。拟议的研究将补充目标1，并允许C8的结构特征与功能相关。将对1 MACPF中参与结合C82和C9的位点以及C82的MACPF片段（2 MACPF）中参与结合C81和C5 b的位点进行鉴定和表征。还将检查C83与C81结合的功能意义。3)确定人类C9的结构-功能关系。该目标将集中于表征C9上的C81结合位点以及介导C9-C9相互作用和聚C9形成的位点。聚C9形成的机制也将被检查。将尝试生产重组C9 MACPF片段，用于C9结合相互作用的详细研究。还将努力使C9结晶以用于结构测定。该项目的重点是一组人类血液蛋白质，它们相互作用并形成复合物，破坏细胞膜并有助于杀死病原体，例如细菌。然而，它也可以改变人体细胞膜，并有助于炎症性疾病的发病机制。这些蛋白质的相互作用将被详细研究，结果可能导致控制复合物形成和功能的药物的开发。

DESCRIPTION (provided by applicant): The objective of this project is to obtain a detailed understanding of the formation and function of human C5b-9, the cytolytic complex of complement referred to as the "membrane attack complex" or MAC. The MAC is a product of the innate immune system. The MAC provides protection against bacterial infection but it also contributes to the pathogenesis of inflammatory diseases. Assembly of the MAC occurs on target cell membranes by a poorly understood mechanism of protein-protein and protein-lipid interactions between complement components C5b, C6, C7, C8 and C9. This project will provide new insight into the molecular basis of these interactions and lay the foundation for designing inhibitors of MAC formation. Specific aims are: 1) Determine the crystal structure of human C8, a 151-kDa protein composed of three genetically distinct subunits, C81, C82, and C83, which are arranged as a disulfide-linked C81-3 heterodimer that is noncovalently associated with C82. Recombinant forms of the central 40-kDa MACPF segment of C81 (1MACPF), and 1MACPF linked to C83 (1MACPF-3), have been crystallized and their structures partially solved. These data will facilitate solving the crystal structure of whole C8 for which X-ray diffraction data (3.0 E resolution) have recently been obtained. 2) Identify and characterize functionally important binding sites on C8. Proposed studies will complement Aim 1 and allow C8 structural features to be correlated with function. Sites in 1MACPF that are involved in binding C82 and C9, and those in the MACPF segment of C82 (2MACPF) which are involved in binding C81 and C5b, will be identified and characterized. The functional significance of C83 binding to C81 will also be examined. 3) Identify structure-function relationships in human C9. This aim will focus on characterizing the C81 binding site on C9 and the site(s) that mediates C9-C9 interaction and polyC9 formation. The mechanism of poly C9 formation will also be examined. Attempts will be made to produce a recombinant C9 MACPF segment to be used for detailed studies of C9 binding interactions. Efforts will also be made to crystallize C9 for structure determination. NARRATIVE This project focuses on a group of human blood proteins that interact and form a complex which disrupts cell membranes and contributes to the killing of pathogenic organisms, e.g. bacteria. However, it can also alter human cell membranes and contribute to the pathogenesis of inflammatory diseases. The interaction of these proteins will be studied in detail and the results could lead to the development of drugs that control the formation and function of the complex.