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Structure-function analysis of the active sites of complement receptor type 1

M Krych1, R Hauhart, J P Atkinson

  • 1Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

The Journal of Biological Chemistry
|May 16, 1998
PubMed
Summary

Mutagenesis of complement receptor type 1 (CR1) identified key residues for iC3/C3b and C4b binding. A CR1 derivative with therapeutic potential as a complement inhibitor was developed.

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biochemistry

Background:

  • Complement receptor type 1 (CR1), also known as CD35, plays a crucial role in regulating the complement system.
  • CR1 contains multiple homologous complement control protein (CCP) repeats, each potentially contributing to its function.
  • Understanding the structure-function relationship of CR1's active sites is essential for developing targeted therapies.

Purpose of the Study:

  • To characterize two functionally distinct homologous sites within CR1 using homologous substitution mutagenesis.
  • To elucidate the role of ionic forces and specific amino acid residues in the binding of CR1 to complement fragments iC3/C3b and C4b.
  • To identify potential therapeutic candidates for complement inhibition based on CR1 structure-function analysis.

Main Methods:

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  • Homologous substitution mutagenesis was performed on two CR1 derivatives, each containing a single active site.
  • Mutated proteins were analyzed for their interaction with iC3/C3b and C4b.
  • Cofactor activity and binding affinities were assessed to determine functional changes.
  • Epitopes for monoclonal antibodies were mapped to understand their functional impact.

Main Results:

  • Modulating charge within the CR1 active sites significantly altered interactions with iC3/C3b and C4b, confirming the role of ionic forces.
  • Substitution of Aspartic acid (Asp) with Asparagine (Asn) in CCP 2 yielded a CR1 derivative with native-like activity but reduced size, indicating therapeutic potential.
  • Residues located between CCPs were found to influence protein activity, likely by affecting CCP positioning.
  • Distinct binding domains for iC3/C3b and C4b were identified, although they overlap.
  • Cofactor activity and binding are generally correlated but separable, suggesting distinct functional mechanisms.
  • Monoclonal antibody binding sites were mapped, explaining their blocking or enhancing effects on CR1 function.

Conclusions:

  • Specific amino acid substitutions can generate CR1 derivatives with therapeutic potential as complement inhibitors.
  • The structural organization of CCP modules and inter-CCP residues is critical for CR1 function.
  • CR1 exhibits overlapping yet distinct binding sites for different complement fragments.
  • CR1's binding and cofactor activities are separable, offering avenues for fine-tuning its function.
  • Understanding CR1's active sites and antibody interactions provides a comprehensive basis for complement system modulation.