Three residues at the interface of factor XI (FXI) monomers augment covalent dimerization of FXI.
BACKGROUND
Human plasma factor XI is a homodimer, with each monomer comprising a catalytic domain and four homologous 'apple' domains. The monomers bind to each other through non-covalent bonds and through a disulfide bond between Cys321 residues in apple 4 domains.


OBJECTIVE
To identify residues essential for dimerization in the FXI monomer interface.


METHODS
Specificity-determining residues in apple 4 domains were sought by sequence alignment of FXI and prekallikrein apple domains in different species. Specific residues identified in apple 4 domains were mutagenized and expressed in baby hamster kidney (BHK) cells for evaluation of their effect on FXI dimerization, analyzed by non-reduced sodium dodecylsulfate polyacrylamide gel electrophoresis and size-exclusion chromatography.


RESULTS
Among the 19 residues of the FXI monomer interface, Leu284, Ile290 and Tyr329 were defined as specificity-determining residues. Substitutions of these residues or pairs of residues did not affect FXI synthesis and secretion from transfected BHK cells, but did impair dimerization, despite the presence of cysteine at position 321. The double mutant 284A/290A yielded predominantly a monomer, whereas all other single or double mutants yielded monomers as well as disulfide-bonded dimers.


CONCLUSIONS
The data suggest that Leu284, Ile290 and Tyr329 in the interface of FXI monomers are essential for forming non-covalently bonded dimers that facilitate formation of a disulfide-bonded stable FXI dimer.
Zucker
M
M
The Amalia Biron Research Institute of Thrombosis and Haemostasis, Chaim Sheba Medical Centre, Tel-Hashomer, Israel.
Zivelin
A
A
Landau
M
M
Rosenberg
N
N
Seligsohn
U
U
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002850:Chromatography, Gel; D006224:Cricetinae; D018076:DNA, Complementary; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005172:Factor XI / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005172:Factor XI