Apple four in human blood coagulation factor XI mediates dimer formation

Area of Science:

• Biochemistry
• Molecular Biology
• Hematology

Background:

• Human blood coagulation factor XI is a homodimer with subunits containing apple domains and a serine protease region.
• A disulfide bridge involving Cys321 links the two subunits of factor XI.

Purpose of the Study:

• To investigate the role of Cys321 in factor XI dimerization.
• To determine if the fourth apple domain mediates subunit association.

Main Methods:

• Site-directed mutagenesis of Cys321 to Serine.
• Expression and purification of recombinant factor XI.
• SDS-PAGE and gel filtration chromatography to assess protein structure.
• Creation of a fusion protein (apple4-tPA) to test the dimerization potential of the apple4 domain.

Main Results:

• Recombinant factor XI with Cys321 mutated to Serine migrated as a monomer under SDS-PAGE but as a dimer under native conditions.
• The disulfide bond is not essential for factor XI dimer formation.
• The apple4 domain, when fused to tissue plasminogen activator (tPA), induced dimerization of the fusion protein.
• The apple3 domain did not promote dimerization.

Conclusions:

• The fourth apple domain of factor XI is responsible for mediating the dimerization of its subunits.
• The interchain disulfide bond involving Cys321 is not essential for factor XI dimer formation.