The long-term objective of this project is to gain a detailed understanding of the mechanism of assembly 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 and the terminal components C6, C7, C8 and C9. Among the latter, C6, C7, the C8-alpha and C8-beta subunits, and C9 comprise a unique family of proteins with highly conserved gene structures, amino acid sequences and a common modular design. The C8-gamma subunit is unrelated and is a member of the lipocalin family of widely distributed proteins that bind small hydrophobic ligands. Because of its central role in MAC assembly, its multi-subunit structure and its multiple interactions, studies will focus primarily on the structure and function of human C8.
Specific aims are to: (1) identify the C8b and C9 binding sites in C8a; (2) identify the segment of C8a that mediates intracellular recognition and binding of C8g and formation of the C8a-g dimer; (3) identify the C5b-7 and C8a-g binding sites in C8b; (4) determine the crystal structure of C8g and extend ongoing studies of its ligand-binding properties and function. Experiments will use recombinant (r) forms of human C8a-g, C8a, C8b and C8g. Binding sites will be identified using truncated mutants and chimeras of rC8a and rC8b in which segments are systematically exchanged and the products analyzed for a corresponding exchange of function. Because of their likely role in mediating protein-protein interactions during MAC assembly, studies will focus primarily on the modules conserved in each protein. Other experiments will attempt to crystallize C8, C8a-g, C8b and rC8a. Diffraction-quality crystals of rC8g have been produced and the structure will be completed. The function of C8g and identity of its natural ligand are unknown. Experiments will search for possible ligands as well as investigate the possibility that C8g binds inflammatory mediators and thus may inhibit proinflammatory responses. Other experiments will examine its role in bacterial killing. Proposed studies of human C8 will provide new insight into the mechanism by which all the terminal components interact and thereby facilitate the design and development of therapeutically useful analogues of MAC and regulators of MAC lytic and stimulatory functions. Because the MAC family of proteins is unique in terms of structure and function, information obtained will also contribute to an understanding of protein-protein interactions in general.
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