The purpose of the proposed research is to investigate basic mechanisms of activation of the human alternative pathway and to learn in molecular detail how the proteins recognize potential pathogens and distinguish them from host cells and tissues. The work addresses four questions central to the biological functions of the pathway. First, how does factor H recognize human cells and tissues and protect them from damage by the continuously activating alternative pathway. Work will focus on the recently described site on factor H centered on the 13th SCR domain which interacts with polyanions prevalent on human cells and tissues. Site-directed mutagenesis of this site will be guided by the NMR-derived structure of SCR domains and the mutagenized proteins will be expressed and examined for functional changes. Second, discrimination between weak and strong activators (i.e. nonvirulent and virulent organisms) is not well understood, but is medically important. Two sites on C3b have been identified which appear to regulate the rate and extent of activation. The specificity and structure of these sites will be examined. Third, the mechanism of thioester reformation following scission of the thioester by ammonia will be investigated. A conformational intermediate has been trapped at low temperature which can refold at higher temperatures to give native C3 with spontaneous thioester reformation between Cys-988 and Gln-991. Experiments are proposed to reveal the chemistry involved, the energy requirements and the presence of similar conformational intermediates for C4 and alpha2M. Fourth, little is known about the structure and function of the C5 convertase of the alternative pathway. Because a widely accepted theory about the structure of C5 convertases has recently been disproven and almost nothing is known about its catalytic activity, the structure and function of this critical enzyme will be investigated.
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