LOCALIZATION OF THE BINDING-SITE ON PLASMA KALLIKREIN FOR HIGH-MOLECULAR-WEIGHT KININOGEN TO BOTH APPLE-1 AND APPLE-4 DOMAINS OF THE HEAVY-CHAIN

The C-terminal end of the heavy chain of human plasma prekallikrein or kallikrein contains a binding site for high-molecular-weight kininogen, the nonenzymatic procofactor of contact activation. To further map this binding site, a series of overlapping peptides were synthesized. The amount of kallikrein that bound to kininogen-coated microtiter plate wells in the presence of increasing concentrations of each peptide was determined by kallikrein amidolytic activity. A peptide encompassing Lys(266)-Gly(295) of kallikrein, conformationally constrained by a disulfide bond, displayed the lowest K-d value (similar to 67 mu M). The linear peptide, Leu(262)-Gly(295), displayed lower affinity (129 mu M). N-terminal or C-terminal truncation/extension peptides of this sequence diminished binding activity. Since the closely related protein, factor XI, has been shown to bind kininogen, a kallikrein-based peptide (Phe(56)-Gly(86)) homologous to the binding domain of FXI, was examined and found to possess less, but significant, binding affinity for kininogen (K-d 530 mu M). Isothermal titration calorimetry was used to assess binding between the kallikrein-based peptides and a peptide encompassing the kallikrein binding domain in kininogen (Ser(565)- Lys(595)). Leu(262)-Gly(295) possesses potent binding activity (K-d 52 mu M), while Phe(56)-Gly(86) displays poorer binding activity (K-d 400 mu M). These interactions are endothermic and entropically favored, suggesting that a conformational rearrangement takes place upon binding. We conclude that the binding site for kininogen within prekallikrein is composed of discontinuous linear segments that form a contiguous surface in the folded protein. (C) 1994 Academic Press, Inc.