Structure/Function of Terminal Complement Proteins

描述（由申请人提供）：本项目的长期目标是了解人C5 b-9的形成和功能的分子细节，补体的细胞溶解复合物被称为“膜攻击复合物”或MAC。MAC由C5 b、C6、C7、C8和C9组成。在这些组分中，C6、C7、C8 α和C8 β亚基以及C9是同源的，而C8 γ具有作为补体系统中唯一的脂质运载蛋白的区别。该项目将重点放在C8作为一个模型系统，以确定MAC蛋白质内的结构-功能关系。重点将放在C8 γ和它的可能性有一个以前未认识到的MAC的形成和功能的作用。具体目标是：（1）表征C8 γ的配体结合特性。C8 γ晶体结构显示出典型的脂质运载蛋白折叠，具有一个独特的结合位点，用于一种尚未鉴定的小分子。研究将集中在脂肪酸和相关化合物作为潜在的配体，以确定C8 γ是否具有结合膜脂质的能力，例如甘油磷脂和LPS，或可能在MAC附近释放的可溶性促炎分子;（2）测试C8 γ通过与膜相关脂质直接相互作用增强MAC活性的假设。将制备配体结合受限的突变体，并就增强MAC溶血和杀菌活性的能力的保留/丧失进行表征。阳性结果表明C8 γ可能参与诱导有核细胞中MAC介导的反应;（3）鉴定C8中功能重要的结合位点。C8 γ上结合C8 α的位点将使用C8 γ及其结构同源物NGAL的嵌合构建体来鉴定，并测试这些构建体的C8 α结合。结合中间MAC复合物C5 b-7的C8 β区域将使用C8 α/C8 β嵌合体和C8 β缺失突变体来确定模块的作用;（4）产生MAC蛋白的片段用于结晶和结构研究。C8 α和C8 β的片段将通过重组和血清来源的C8的蛋白水解消化产生。前一种方法将强调中央MACPF片段，因为它很可能是可以表达的自折叠结构域。后一种方法将利用规模优势。完成后，结果将提供新的见解MAC蛋白相互作用和靶细胞膜的机制。这些信息将有助于设计和开发治疗上有用的MAC类似物和MAC裂解和刺激功能的调节剂。由于MAC蛋白及其功能的独特性，所获得的信息将有助于理解蛋白质的结构-功能关系。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand molecular details of the formation and function of human C5b-9, the cytolytic complex of complement referred to as the "membrane attack complex" or MAC. MAC is composed of C5b, C6, C7, C8 and C9. Among these components, C6, C7, the C8alpha and C8beta subunits, and C9 are homologous whereas C8gamma has the distinction of being the only lipocalin in the complement system. This project will focus on C8 as a model system to identify structure-function relationships within the MAC proteins. Emphasis will be on C8gamma and the possibility it has a previously unrecognized role in MAC formation and function. Specific aims are: (1) to characterize the ligand binding properties of C8gamma. The C8gamma crystal structure displays a typical lipocalin fold with a distinct binding site for an as yet unidentified small molecule. Studies will focus on fatty acids and related compounds as potential ligands to determine if C8gamma has the capacity to bind membrane lipid, e.g. glycerophospholipids and LPS, or possibly soluble proinflammatory molecules released in the vicinity of the MAC; (2) to test the hypothesis that C8gamma enhances MAC activity through direct interaction with membrane-associated lipid. Mutants in which ligand binding is restricted will be prepared and characterized with respect to retention/loss of ability to enhance MAC hemolytic and bactericidal activities. Positive results would suggest C8gamma may be involved in inducing MAC-mediated responses in nucleated cells; (3) to identify functionally important binding sites in C8. The site on C8gamma that binds C8alpha will be identified using chimeric constructs of C8gamma and its structural homologue NGAL, and testing these for C8alpha binding. The region of C8beta which binds the intermediate MAC complex C5b-7 will use C8alpha / C8beta chimeras and C8beta deletion mutants to determine the role of the modules; (4) to produce fragments of the MAC proteins for crystallization and structural studies. Segments of C8alpha and C8beta will be produced recombinantly and by proteolytic digestion of serum-derived C8. The former approach will emphasize the central MACPF segment because it is likely to be self-folding domain that can be expressed. The latter approach will take advantage of scale. When complete, results will provide new insight into mechanisms by which the MAC proteins interact with each other and the target cell membrane. Such information will facilitate design and development of therapeutically useful analogues of MAC and regulators of MAC lytic and stimulatory functions. Because of the uniqueness of the MAC proteins and their function, information obtained will contribute to an understanding of protein structure-function relationships in general.