BINDING OF HIGH-MOLECULAR-MASS KININOGEN TO THE APPLE-1 DOMAIN OF FACTOR-XI IS MEDIATED IN PART BY VAL(64) AND ILE(77)

We have previously demonstrated the presence of a binding site for hig h-molecular-mass kininogen (HK), spanning residues Val(59)-Lys(83), in , the first Apple (Al) domain in the heavy-chain region of factor XI. We have now prepared conformationally constrained synthetic peptides a nd recombinant Al domain (rAl) constructs to identify the specific ami no acid residues that constitute the HK-binding site. Expression of th e Al domain (Glu(1)-Ser(90)) was achieved in a bacterial expression sy stem following PCR amplification of the Al domain from factor XI cDNA and ligation into an expression plasmid. The rAl inhibited factor XI b inding to HK [K-i similar to (2-3) x 10(-7) M] in a manner indistingui shable from purified factor XI, indicating that all the information ne cessary for binding HK is contained within the Al domain, To identify specific amino acid residues involved in binding HK, conformationally constrained peptides were synthesized containing conservative amino ac id substitutions at residues suspected to contain side chains involved in binding, including Val(64) --> Ala, Glu(66) --> Ala, Arg(73) --> A la and Ile(77) --> Ala. Because normal results were obtained with all peptides with the exception of Val(64) --> Ala and Ile(77) --> Ala, wh ich failed to compete normally with factor XI for binding to HK, we pr epared two mutant rAl domains (Val(64) --> Ala and Ile(77) --> Ala) by PCR-based site-directed mutagenesis, both of which exhibited diminish ed capacity to inhibit factor XI binding to HK. Competition studies wi th prekallikrein (PK) and a PK-dependent synthetic peptide suggested t hat PK and factor XI have a common surface in the Al domain for bindin g HK of which Val(64) is a part. We conclude that the binding of facto r XI to HK is mediated at least in part by Val(64) and Ile(77) in the Al domain of factor XI.