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Human C4b-binding protein has overlapping, but not identical, binding sites for C4b and streptococcal M proteins
A M Blom1, K Berggârd, J H Webb
1Department of Clinical Chemistry, Lund University, University Hospital Malmö, Malmö, Sweden.
Abstract:
Many strains of Streptococcus pyogenes bind C4b-binding protein (C4BP), an inhibitor of complement activation. The binding is mediated by surface M proteins in a fashion that has been suggested to mimic the binding of C4b. We have previously shown that a positively charged cluster at the interface between complement control protein domains 1 and 2 of C4BP alpha-chain is crucial for the C4b-C4BP interaction. To extend this observation, and to investigate the interaction with M proteins, we constructed and characterized a total of nine mutants of C4BP. We identified a key recognition surface for M proteins that overlaps with the C4b binding site because substitution of R64 and H67 by Gln dramatically reduces binding to both ligands. However, the analysis of all mutants indicates that the binding sites for C4b and M proteins are only overlapping, but not identical. Furthermore, M proteins were able to displace C4BP from immobilized C4b, whereas C4b only weakly affected binding of C4BP to immobilized M proteins. We found that the molecular mechanisms involved in these two interactions differ because the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b binding. In addition, six mAbs directed against the alpha-chain interfered with C4b-C4BP interaction, whereas only two of them efficiently inhibited binding of C4BP to M proteins. Collectively, our results suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional noncovalent forces cause tight binding of C4BP to streptococcal M proteins.
Insights
Streptococcus pyogenes M proteins bind C4b-binding protein (C4BP) at a site overlapping but distinct from the C4b binding site. This interaction involves different molecular mechanisms than C4b binding, suggesting unique forces mediate C4BP
Area of Science:
- Immunology
- Microbiology
- Complement System Biology
Background:
- Streptococcus pyogenes utilizes surface M proteins to bind C4b-binding protein (C4BP), a key regulator of complement activation.
- Previous research identified a crucial positively charged cluster in C4BP's alpha-chain (domains 1 and 2) for C4b interaction.
Purpose of the Study:
- To investigate the interaction between C4BP and Streptococcus pyogenes M proteins.
- To delineate the binding sites and molecular mechanisms involved in C4BP-C4b and C4BP-M protein interactions.
Main Methods:
- Construction and characterization of nine C4BP mutants.
- Analysis of binding affinities and displacement assays between C4BP, C4b, and M proteins.
- Salt sensitivity and monoclonal antibody (mAb) inhibition studies.
Main Results:
- A key M protein recognition surface on C4BP overlaps with the C4b binding site, with specific mutations (R64, H67) reducing binding to both ligands.
- Binding sites are overlapping but not identical; M proteins can displace C4BP from C4b, but not vice versa.
- C4BP-M protein binding is salt-insensitive, unlike salt-sensitive C4BP-C4b interactions.
- Monoclonal antibodies differentially inhibited C4BP binding to C4b versus M proteins.
Conclusions:
- C4BP binding to C4b is primarily driven by electrostatic interactions.
- Additional noncovalent forces contribute to the tight binding of C4BP to streptococcal M proteins.
- The distinct binding mechanisms highlight the complex interplay between complement regulation and bacterial evasion strategies.