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Molecular engineering of an efficient four-domain DAF-MCP chimera reveals the presence of functional modularity in RCA proteins

Significance Complement regulation is critical for protecting the body’s own cells from homologous complement. This function is primarily mediated by the regulators of complement activation (RCA) proteins through two functional mechanisms termed decay-accelerating activity (DAA) and cofactor activity (CFA). Here, we show the presence of functional modularity in the RCA proteins. We demonstrate that the basic functional blocks for DAA and CFA in these proteins are formed by only two domains and that these are arranged in a specific order. Furthermore, we show that linking functional blocks of the decay-accelerating factor and membrane cofactor protein—which possess DAA and CFA, respectively—and adding C-terminal domains for enhancing avidity toward C3b/C4b results in a regulator molecule with dual activity. The complement system is highly efficient in targeting pathogens, but lack of its apposite regulation results in host-cell damage, which is linked to diseases. Thus, complement activation is tightly regulated by a series of proteins, which primarily belong to the regulators of complement activation (RCA) family. Structurally, these proteins are composed of repeating complement control protein (CCP) domains where two to four successive domains contribute to the regulatory functions termed decay-accelerating activity (DAA) and cofactor activity (CFA). However, the precise constitution of the functional units and whether these units can be joined to form a larger composition with dual function have not been demonstrated. Herein, we have parsed the functional units for DAA and CFA by constructing chimeras of the decay-accelerating factor (DAF) that exhibits DAA and membrane cofactor protein (MCP) that exhibits CFA. We show that in a four-CCP framework, a functional unit for each of the regulatory activities is formed by only two successive CCPs wherein each participates in the function, albeit CCP2 has a bipartite function. Additionally, optimal activity requires C-terminal domains that enhance the avidity of the molecule for C3b/C4b. Furthermore, by composing a four-CCP DAF-MCP chimera with robust CFA (for C3b and C4b) and DAA (for classical and alternative pathway C3 convertases), named decay cofactor protein, we show that CCP functional units can be linked to design a dual-activity regulator. These data indicate that the regulatory determinants for these two biological processes are distinct and modular in nature.