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

Abstract

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. complement | complement regulation | RCA proteins | decay-accelerating factor | membrane cofactor protein Author contributions: H.

The imitation game: a viral strategy to subvert the complement system

“…A similar conclusion was also drawn following the functional analyses of domain swap mutants of VCP and Kaposica, wherein individual domains of these viral regulators were swapped with the homologous domains of DAF or MCP [121,123]. A recent study on DAF and MCP chimera showed that domains 1-2 (homologous domains in DAF are CCP 2-3) and 2-3 also serve as functional modules for DAA and CFA in these proteins [125]. It can, therefore, be concluded that vRCAs are true homologs of huRCA proteins.…”

“…Fourth, regulatory determinants in RCAs are conserved evolutionarily. The 'functional blocks' for CFA and DAA in the vRCAs were shown to be conserved in the huRCAs too, and it led to the construction of a dual-activity human DAF-MCP chimera [125]. Besides, the functional motifs [139] and hotspots [118,123] are also evolutionarily conserved.…”

“…The transient nature of binding suggested that the fast recycle rate of the viral regulators for C3 convertases helps them to inactivate multiple convertases in a relatively short time. It is important to note that DAF and MCP also display similar binding mechanisms [113,124,125]. Recent surface plasmon resonance data on DAF-MCP chimeras showed that mutant with slower off-rate has significantly lower DAA activity [125].…”

“…It is important to note that DAF and MCP also display similar binding mechanisms [113,124,125]. Recent surface plasmon resonance data on DAF-MCP chimeras showed that mutant with slower off-rate has significantly lower DAA activity [125]. Thus, the transient binding mechanism seems to be a general mode of target binding mechanism adopted by the RCA proteins.…”

“…The spatially conserved motifs are marked with a shaded background: motif 5 (yellow), motif 3 (light pink), motif 1 (dark green), motif 2 (light green) and motif 4 (salmon red). The interface of regulators with C3b is marked with black underlines, and that with factor I and Bb is marked with red and blue underlines, respectively [112,113,125,132]. Lossof-function mutations are highlighted with different coloured bold fonts: loss in CFA, red font; loss in DAA, turquoise font; and loss in CFA and DAA, black font.…”