Assembly and enzymatic properties of the catalytic domain of human complement protease C1r

The catalytic properties of C1r, the protease that mediates activation of t he C1 complex of complement, are mediated by its C-terminal region, compris ing two complement control protein (CCP) modules followed by a serine prote ase (SP) domain. Baculovirus-mediated expression was used to produce fragme nts containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only t he second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, where as all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that Clr activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dim ers (5.5-5.6 S), and all CCP2-SP fragments were monomers (3.2-3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mu tants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielde d enzymes with Cls-cleaving activities similar to their active wild-type co unterparts. Clr and its activated fragments all cleaved Cls, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 i s not involved in Cls recognition.