Human complement is a group of proteins that form the principal effector arm of the humoral immune system and plays an important role in defending the host against invading pathogens. Complement- mediated inflammation although necessary for efficient elimination of microorganisms, also can lead to the damage of host tissue. Inappropriate activation of complement occurs when the inhibition systems are """"""""over- ridden"""""""" leading to acute inflammation and tissue destruction. The long term goal of this project is to delineate at the atomic level the structural determinants for the formation, regulation, and mechanism of action of key enzymes (factor B and C2) in the activation and formation of classical and alternative pathway C3 convertases. We are particularly interested in determining the three dimensional structures of factor ?, factor B and C2 and use this structural information in the design of specific inhibitors. We propose to achieve our goals by pursuing three specific aims: 1) Define structural correlates of factor D: We will approach this by the combination of X-ray crystallography, molecular modeling and protein engineering. Our structural studies will continue on mutants of factor D, pro-factor D and factor D crystallized at different pHs. 2) Structural studies on factor B and C2. This includes expression, purification, crystallization, and structure determination of individual domains and intact factor B and C2. We have cloned and expressed three out of six individual domains. Purification and crystallization of these domains are in progress. 3) Structure based inhibitor designed for factor D, factor B and C2: We have identified a family of inhibitors, co- crystallized three of these and refined the structures. The interactive and iterative process o structure based inhibitor design is in progress. Similar process will be initiated once we determine the structures of the serine protease domains Bb-C and C2a-C. The structural data we propose to collect are necessary for attaining the important goal of pharmacologic control of complement activation.
