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The disulfide bonding pattern in ficolin multimers.

Tomoo Ohashi1, Harold P Erickson

  • 1Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

The Journal of Biological Chemistry
|December 9, 2003
PubMed
Summary

Ficolin multimerization relies on specific cysteine residues. Mutating Cys24 disrupts disulfide bonds, preventing 12-mer formation and forming trimers instead, revealing key structural insights.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Immunology

Background:

  • Ficolins are plasma lectins crucial for innate immunity.
  • They assemble into 12-mers via N-terminal and collagen domains.
  • Disulfide bonds in N-terminal cysteines are hypothesized to mediate multimerization.

Purpose of the Study:

  • To investigate the role of N-terminal cysteines (Cys4, Cys24) in ficolin alpha multimerization.
  • To determine the structural contribution of disulfide bonds to ficolin assembly.
  • To elucidate the mechanism of ficolin 12-mer formation.

Main Methods:

  • Site-directed mutagenesis to create Cys4, Cys24, and Cys4/Cys24 ficolin alpha mutants.
  • Mammalian cell expression and affinity purification of recombinant proteins.
  • Analysis of protein multimerization using glycerol gradient sedimentation and electron microscopy (nondenaturing conditions).
  • Nonreducing SDS-PAGE to assess disulfide-linked covalent structures (denaturing conditions).

Main Results:

  • Wild-type and Cys4 ficolin alpha formed stable 12-mers.
  • Cys24 and Cys4/Cys24 mutants predominantly formed trimers.
  • Nonreducing SDS-PAGE revealed wild-type ficolin formed covalent 12-mers, while mutants yielded dimers and monomers.
  • Cys4-mediated interactions are stable noncovalently, while Cys24-Cys24 interactions require disulfide bonds.

Conclusions:

  • Cys24 residues are essential for forming stable, disulfide-linked trimers into the 12-mer structure.
  • The Cys4 residue contributes to noncovalent interactions stabilizing the multimeric structure.
  • A model proposing symmetric Cys24-Cys24 disulfide bonds as the basis for ficolin multimerization is presented.
  • This finding may extend to the structural basis of collectin multimerization.

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