Structural insights into cofactor activity

Regulators of complement activation (RCA) modulate the innate immune response to avoid self-injury from inadvertent attack. These RCA proteins exert two distinct activities to control complement activity: (1) enhance dissociation of the C3 convertases, (i.e., decay acceleration activity), and/or (2) induce proteolytic degradation of C3b and C4b by Factor I (FI), (i.e., cofactor activity). Cofactor activity is not only important for complement regulation but also critical for invoking adaptive immune responses, such as phagocytosis and B cell stimulation. However, the molecular mechanism of how the regulators and FI work together to cleave C3b/C4b multiple times at distant sites (∼20-Å apart) has remained unclear. Here we provide structural insights into the complex formed by C3b, Factor H (FH) and mature FI, trapping the transient protease:substrate complex by mutating FI's catalytic serine into an alanine. The structure reveals how C3b-FH provides a platform for FI binding and how the serine protease (SP) domain of FI is stabilized by interactions with the substrate and cofactor critical for proteolytic activity. Analysis of interactions between the FI and the regulator reveals several critical residues on the regulator that activate the SP domain. Mutation of corresponding residues in DAF confers cofactor activity. Based on the ternary structure of cofactor-enzyme-substrate complex and the structural chances induced upon C3b cleavages, we propose a mechanistic model for cleavage of the subsequent cleavages sites. Moreover, the model explains why MCP allows FI to cleave C3b only two times, FH two-three times and CR1 three times, yielding different proteolytic fragments of C3b on the cell or particle surface and, hence, causing differential immune responses.