The long-term objective of our proposed research is to delineate at the atomic level the structural determinants responsible for the formation, activation and regulation of two key human complement enzymes, the C3-convertases. Towards this goal, with the help of various biophysical techniques, we completed the structural characterization of factor D and laid foundation for our 'structure based inhibitor design' efforts, resulting in small molecule factor D inhibitors with nanomolar binding affinities. In this proposal, we intend to extend our studies to factor B and C2 that provide the proteolytic machinery for C3-convertases. We determined the crystal structures of the individual C-terminal serine protease and Bb fragment of factor B. This helped us to propose, contrary to previous suggestions, that the co-factor induced transient conformational changes emanating from the middle domain of factor B cannot activate its C-terminal serine protease domain. We hypothesize, in the case of the alternative pathway C3-convertase C3bBb, the zymogen-like catalytic component of Bb undergoes substrate-induced activation, with the assistance of co-factor C3b in substrate binding. We propose to validate this hypothesis by pursuing four Specific Aims: (1) Define the structural correlates of factor B and C2. We have expressed full length factor B and C2 in baculovirus and produced diffraction quality crystals of C2. Structural studies would help us visualize the role of the N-terminal Ba and C2b in the assembly and function of factor B and C2 respectively. (2) Site-directed mutagenesis of unique substitutions and additions in the serine protease domain of factor B would help us to understand their role in the catalytic function and efficiency. The zymogen like serine protease domain needs external help for activation and we would attempt delineate such specific structural requirements. (3) With the help of Surface Plasmon Resonance (BIAcore) and electron microscopy, we would try to resolve the stoichiometry of co-factor C3b and factor B interactions. 4) We will determine the crystal structures of Bb-inhibitor complexes, which would help us to delineate substrate binding sub sites and their specificities, an essential knowledge for future 'structure based inhibitor design1 efforts. We believe that the proposed research will provide structural correlates for achieving our ultimate goal of pharmacological control of human complement.
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