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.
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