The signature feature of mammalian complement is the ability to kill invading cells by puncturing their plasma membranes. This assault is carried out by the Membrane Attack Complex (MAC), which is a >1,000,000 Da protein complex composed of 5 different serum proteins: single copies of complement components C5b, C6, C7, and C8, and 1 or more copies of component C9. MAC assembly begins with the binding of newly generated C5b to C6 near the target cell surface. Progressively larger complexes are then formed by the spontaneous and sequential addition of C7, C8, and finally multiple copies of C9. Very little is known about this self-assembly process, the structure of the fully-assembled MAC or its intermediates, or even of the 3D structures of the individual MAC proteins. The long-term goal of the proposed studies is to obtain a better picture of the MAC: how the individual proteins are arranged in the complex, what forces drive independent soluble serum proteins to aggregate into the membrane-bound MAC, and how this process is regulated.
Our Specific Aims i n this application are to (1) use surface plasmon resonance to describe in detail the binding process at each step in MAC assembly and in binding of the regulatory S-protein, (2) map domain-domain interactions early in MAC assembly by expressing individual domains of C6 and C7 in bacteria and measuring their binding activities, (3) determine the 3D structures of expressed domains by NMR or by X-ray crystallography in collaboration with experts in these technologies, and (4) map the active sites within modules by site-specific mutagenesis. These studies should provide insights into the structures of C6 and C7, and into the protein-protein interactions that take place early in MAC formation. This information should allow us to begin to fashion a molecular model of MAC assembly and structure. These studies have substantial relevance to human disease. While the MAC is best known for its defense against infection by foreign cells, it can attack and injure host cells as well. For example, MAC attack on host cells has been identified as a major contributor to the vascular complications of diabetes and the inflammatory pathogenesis of atherosclerosis. A clearer picture of the MAC will provide insights into the design of drugs that control MAC assembly and injury in these prevalent and devastating human diseases. ? ? ? ?
