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Factor XI homodimer structure is essential for normal proteolytic activation by factor XIIa, thrombin, and factor XIa
Wenman Wu1, Dipali Sinha, Sergei Shikov
1Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
The Journal of Biological Chemistry
|April 29, 2008
Summary
Coagulation factor XI (FXI) homodimerization is crucial for its activation. Specific salt bridges and hydrophobic interactions mediate FXI dimerization, essential for activation by FXIIa and thrombin.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Structure
Background:
- Coagulation factor XI (FXI) is a homodimer critical for blood coagulation.
- The disulfide bond at Cys-321 normally links the two subunits.
- Understanding the non-covalent interactions maintaining FXI's dimeric structure is key.
Purpose of the Study:
- To identify the specific amino acid residues and interactions responsible for FXI homodimer formation.
- To investigate the functional consequences of disrupting FXI dimerization on its activation and activity.
Main Methods:
- Site-directed mutagenesis of FXI, including Cys-321 substitution and mutations at potential interface residues (Lys-331, Glu-287, Ile-290, Leu-284, Tyr-329, His-343).
- Expression of wild-type and mutant FXI in HEK293 cells.
- Biophysical characterization using size exclusion chromatography, analytical ultracentrifugation, and electron microscopy.
- Functional assays to assess FIX activation and FXI activation by FXIIa, thrombin, or autoactivation.
Main Results:
- Mutations disrupting salt bridges (Lys-331, Glu-287) or hydrophobic interactions (Ile-290, Leu-284, Tyr-329) resulted in predominantly monomeric FXI.
- FXI(C321S) and FXI(C321S,H343A) exhibited monomer/dimer equilibrium, while other mutants were stable monomers.
- Monomeric FXIa variants retained the ability to activate FIX but showed impaired activation by FXIIa, thrombin, or autoactivation.
Conclusions:
- Salt bridges between Lys-331 and Glu-287, along with hydrophobic interactions involving Ile-290, Leu-284, and Tyr-329, are essential for FXI homodimer formation.
- The dimeric structure of FXI is indispensable for its efficient proteolytic activation by upstream factors (FXIIa, thrombin) and autoactivation.
- FXI dimerization is not required for its enzymatic activity in activating FIX once FXIa is formed.
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