Molecular determinants of the substrate specificity of the complement-initiating protease, C1r
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The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defence mechanism against pathogens and altered self cells. C1r both auto-activates and subsequently cleaves and activates C1s. Since complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1prime, which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1prime in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modelling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The CCP domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.
Wijeyewickrema, L. C., Yongqing, T., Tran, P. T., Thompson, P., Viljoen, J. E., Coetzer, T. H., Duncan, R. C., Kass, I., Buckle, A. M., & Pike, R. N. (2013). Molecular determinants of the substrate specificity of the complement-initiating protease, C1r. The Journal of Biological Chemistry, 288(22), 15571 - 15580. https://doi.org/10.1074/jbc.M113.451757