Molecular basis of C-reactive protein binding and modulation of complement activation by factor H-related protein 4
Mario Hebecker1, Azubuike I Okemefuna, Stephen J Perkins
1Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute, Jena, Germany.
Insights
Human complement factor H-related protein 4 (CFHR4) specifically binds C-reactive protein (CRP), enhancing complement activation and opsonization. This interaction involves a unique motif on CFHR4, crucial for its function.
Area of Science:
- Immunology
- Complement System
- Protein-Protein Interactions
Background:
- C-reactive protein (CRP) is a pattern recognition molecule that initiates complement activation via the classical pathway upon binding ligands.
- Human complement factor H-related protein 4 (CFHR4) was previously identified as a binding protein for CRP.
Purpose of the Study:
- To elucidate the molecular basis of the interaction between CFHR4 and native CRP.
- To investigate the functional relevance of CFHR4-CRP binding in complement activation.
Main Methods:
- Recombinant expression of CFHR4 fragments to map the CRP binding site.
- Peptide arrays and site-directed mutagenesis to identify key residues involved in binding.
- Homology modeling to visualize the binding site.
- Assays for C3 fragment deposition to measure complement activation.
Main Results:
- The CRP binding site on CFHR4 was localized to the first short consensus repeat (SCR) domain, specifically involving residues 35-41.
- Mutations in this motif significantly reduced CRP binding and subsequent complement activation.
- Sequence comparisons indicated this binding motif is unique to CFHR4 among related proteins.
Conclusions:
- The study reveals the molecular determinants for the specific interaction between CFHR4 and CRP.
- CFHR4 binding to CRP enhances complement activation, suggesting a role in opsonization.
- The findings highlight CFHR4 as a key mediator in CRP-driven immune responses.
Abstract:
C-reactive protein (CRP) is a pattern recognition molecule that binds several microbial and host ligands. Ligand-bound CRP activates the complement system via the classical pathway. Previously, we identified human complement factor H-related protein 4 (CFHR4), a member of the factor H protein family, as a CRP binding protein. Here, we investigated the molecular basis and the functional relevance of the interaction of CFHR4 with native CRP. Using recombinantly expressed CFHR4 fragments, the CRP binding site was localized to the first short consensus repeat (SCR) domain of CFHR4. Peptide arrays identified residues 35-41 of CFHR4 to be involved in CRP binding. Substitutions of the positively charged amino acids of this motif resulted in strongly reduced CRP binding. Sequence comparisons revealed that such a motif is not present in the related SCR6 domain of factor H, or in the homologous domains of the four other CFHR proteins. Homology modelling based on SCR6 of factor H showed that the CRP binding site is surface exposed on SCR1 of CFHR4. CFHR4-bound CRP was able to activate complement, determined by C3 fragment deposition. Recombinant CFHR4 proteins with mutations in the identified binding site showed reduced CRP binding, which in turn resulted in reduced complement activation. In summary, these data reveal the molecular basis of the specific interaction of CFHR4 with native CRP and suggest a role for CFHR4 in enhancing opsonization via CRP binding.
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