Structure/Function of Terminal Complement Proteins

描述（由申请人提供）：该项目的目的是详细了解人体C5b-9的形成和功能，这是一种补体的细胞溶解复合物，称为“膜攻击复合物”或MAC。MAC是先天免疫系统的产物。MAC提供对细菌感染的保护，但它也有助于炎症性疾病的发病机制。MAC的组装是通过补体组分C5b、C6、C7、C8和C9之间的蛋白质-蛋白质和蛋白质-脂质相互作用机制在靶细胞膜上进行的，这一机制目前尚不清楚。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形成抑制剂奠定基础。具体目标是：

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.