Binding of high-molecular-mass kininogen to the Apple 1 domain of factor XI is mediated in part by Val64 and Ile77.
We have previously demonstrated the presence of a binding site for high-molecular-mass kininogen (HK), spanning residues Val59-Lys83, in the first Apple (A1) domain in the heavy-chain region of factor XI. We have now prepared conformationally constrained synthetic peptides and recombinant A1 domain (rA1) constructs to identify the specific amino acid residues that constitute the HK-binding site. Expression of the A1 domain (Glu1-Ser90) was achieved in a bacterial expression system following PCR amplification of the A1 domain from factor XI cDNA and ligation into an expression plasmid. The rA1 inhibited factor XI binding to HK [Ki approximately (2-3) x 10(-7) M] in a manner indistinguishable from purified factor XI, indicating that all the information necessary for binding HK is contained within the A1 domain. To identify specific amino acid residues involved in binding HK, conformationally constrained peptides were synthesized containing conservative amino acid substitutions at residues suspected to contain side chains involved in binding, including Val64-->Ala, Glu66-->Ala, Arg73-->Ala and Ile77-->Ala. Because normal results were obtained with all peptides with the exception of Val64-->Ala and Ile77-->Ala, which failed to compete normally with factor XI for binding to HK, we prepared two mutant rA1 domains (Val64-->Ala and Ile77-->Ala) by PCR-based site-directed mutagenesis, both of which exhibited diminished capacity to inhibit factor XI binding to HK. Competition studies with prekallikrein (PK) and a PK-dependent synthetic peptide suggested that PK and factor XI have a common surface in the A1 domain for binding HK of which Val64 is a part. We conclude that the binding of factor XI to HK is mediated at least in part by Val64 and Ile77 in the A1 domain of factor XI.
Seaman
F S
FS
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140.
Baglia
F A
FA
Gurr
J A
JA
Jameson
B A
BA
Walsh
P N
PN
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D005172:Factor XI / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000378:metabolism*; D007704:Kininogens / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014633:Valine / Q000378:metabolism*
D007704:Kininogens; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007532:Isoleucine; D018698:Glutamic Acid; D012694:Serine; D005172:Factor XI; D014633:Valine