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 Lys266-Gly295 of kallikrein, conformationally constrained by a disulfide bond, displayed the lowest Kd value (∼67 μM). The linear peptide, Leu262-Gly295, displayed lower affinity (129 μM). 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 (Phe56-Gly86) homologous to the binding domain of FXI, was examined and found to possess less, but significant, binding affinity for kininogen (Kd 530 μM). Isothermal titration calorimetry was used to assess binding between the kallikrein-based peptides and a peptide encompassing the kallikrein binding domain in kininogen (Ser565- Lys595). Leu262-Gly295 possesses potent binding activity (Kd 52 μM), while Phe56-Gly86 displays poorer binding activity (Kd 400 μM). 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.