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Identification of residues within the 727-767 segment of human complement component C3 important for its interaction
1Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Insights
This study identifies key charged residues in the C3b alpha-chain segment (727-767) crucial for interactions with complement regulators factor H and CR1. Mutations reveal distinct binding requirements for factor H, CR1, and membrane cofactor protein (MCP).
Area of Science:
- Complement system biology
- Protein-protein interactions
- Immunology
Background:
- The 727-767 segment of the C3b alpha'-chain is implicated in interactions with complement regulators factor B, factor H, and complement receptor 1 (CR1).
- Previous studies identified Glu-736 and Glu-737 as important for C3b binding to factor B and CR1, but not factor H.
Purpose of the Study:
- To investigate the role of charged residues within the remainder of the 727-767 segment (738-767) in the interaction of C3b/C3(H2O) with factor H, CR1, and membrane cofactor protein (MCP).
- To elucidate the specific binding interfaces and cofactor activities involved in complement regulation.
Main Methods:
- Site-directed mutagenesis was used to create mutant C3(H2O) molecules within the 738-767 segment.
- A cofactor-dependent factor I cleavage assay was employed as a surrogate binding assay to assess interactions with factor H, CR1, and MCP.
- Analysis of mutant interactions with soluble CR1 (sCR1) differentiated cofactor activity for different cleavage steps.
Main Results:
- Glu-744 and Glu-747 are critical for C3(H2O) interaction with factor H.
- Two classes of residues were identified for CR1 cofactor activity: one for initial cleavages (Glu-736/Glu-737, Glu-747, Glu-754/Asp-755) and another for the third cleavage (Glu-744, Lys-757/Glu-758).
- Mutations did not affect C3(H2O) interaction with membrane cofactor protein (MCP), suggesting distinct binding mechanisms.
Conclusions:
- The findings highlight the importance of negatively charged residues in the C3b alpha'-chain segment for interactions with factor H and CR1.
- CR1 binding involves a broader region of the 727-767 segment compared to factor H, with distinct requirements for different cleavage events.
- Membrane cofactor protein (MCP) likely binds C3b/C3(H2O) through a different mechanism than factor H and CR1, despite functional similarities.
Abstract:
Mapping approaches employing blocking antibodies and synthetic peptides have implicated the 727-767 segment at the NH2 terminus of C3b alpha'-chain as contributing to the interactions with factor B, factor H, and CR1. Our previous mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C3b to factor B and CR1 but not factor H. We have now extended the charged residue mutagenic scan to cover the remainder of the segment (738-767) and have assessed the ability of the C3b-like C3(H2O) form of the mutant molecules to interact with factor H, CR1, and membrane cofactor protein (MCP) using a cofactor-dependent factor I cleavage assay as a surrogate binding assay. We have found that the negatively charged side chains of Glu-744 and Glu-747 are important for the interaction between C3(H2O) and factor H, a result in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545-552) which implicated residues within the 744-754 segment in H binding. The interactions of the mutants with soluble CR1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of alpha-chain. These are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-744 and the Lys-757/Glu-758 pairing. The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribute basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wider portion of the 727-767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction between soluble MCP and C3(H2O), indicating that despite its functional homology to H and CR1, MCP differs in its mode of binding to C3b/C3(H2O).