Thermodynamic and structural studies on the formation of the C3 convertase

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DESCRIPTION (provided by applicant): Among the complement pathways, the alternative pathway plays a predominant role in amplification of effector functions in the complement system and has been considered a key target for complement therapeutic interventions. Its activation is initiated when factor B binds to "C3b-like C3" (C3(H2O)). Factor D then cleaves factor B into Ba and Bb and the resulting complex, C3(H20)Bb, serves as the initial C3 convertase capable of cleaving C3 to C3b. The catalytic subunit of the enzyme complex is located in the Bb fragment. The newly generated C3b, as well as any C3b that is generated through the activation of the classical or lectin pathway, can bind another molecule of B and, after factor D cleavage, the alternative pathway C3bBb convertase is established. Although biochemical details of the interaction between C3 and factor B have been accumulated over the past three decades, a more detailed characterization of this binding process is essential for the understanding of its physiological relevance and its implications for C3 convertase formation and function. The long-term goal of this proposal is to identify the structural determinants associated with the formation of C3 convertase. The proposed study will involve a detailed structural and functional analysis associated with the formation of the alternative pathway C3 convertase, by studying the interaction between the human C3(H2O), C3b and the complement proteins factor B and its fragments, Bb and Ba. Protein crystallography, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and hydrogen exchange/mass spectrometry (HDX-MS) will be used to assess the molecular forces that impart specificity and recognition to the interactions between C3 and factor B. The functional sites of the interacting molecules will be mapped using HDX-MS, crystallization, computational methods, and site-directed mutagenesis. The proposed studies are designed to provide basic information on the structural features of CS-factor B interactions as they relate to complement C3 convertase functions. These studies should provide insight into how the complement C3 convertase is formed and also assist in the design of specific inhibitors that may have important medical applications.