Mapping of the high molecular weight kininogen binding site of prekallikrein. Evidence for a discontinuous epitope formed by distinct segments of the prekallikrein heavy chain

Prekallikrein, a glycoprotein involved in contact phase activation, circulates in plasma in the form of a binary complex with high molecular weight kininogen (H-kininogen). The binding to H-kininogen is mediated by the prekallikrein heavy chain consisting of four repetitive domains, A1-A4. To define more precisely the region(s) involved in kininogen binding, we have employed an affinity cross-linking strategy with a synthetic peptide of 31 residues which mimics the prekallikrein binding site of H-kininogen. Cross- linking of the radiolabeled peptide to (pre)kallikrein revealed a binding segment in the NH ₂-terminal portion of the prekallikrein heavy chain; another binding segment was located in the COOH-terminal part of the heavy chain. The latter binding segment is harbored by a previously identified fragment of the kallikrein heavy chain involved in H-kininogen binding (Page, J. D., and Colman, R. W. (1991) J. Biol. Chem. 266, 8143-8148). Chemical cleavage of the heavy chain cross-linked with the radiolabeled peptide mapped the NH ₂-terminal binding segment to 60 residues (positions 53-112) of A1. Synthesis of a peptide (positions 56-86) and development of specific antibodies to this peptide narrowed down the kininogen binding segment to 31 residues of the center portion of A1. This NH ₂-terminal segment is equivalent to a kininogen binding site previously identified in factor XI (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1992) J. Biol. Chem. 267, 4247-4252). We conclude that prekallikrein exposes at least two segments on its heavy chain portion which form a continuous surface thereby facilitating the intimate binding of the zymogen to its nonenzymatic cofactor, H- kininogen.