CONFORMATIONAL-ANALYSIS OF SYNTHETIC PEPTIDES ENCOMPASSING THE FACTOR-XI AND PREKALLIKREIN OVERLAPPING BINDING DOMAINS OF HIGH-MOLECULAR-WEIGHT KININOGEN

High molecular weight kininogen, a plasma glycoprotein, circulates as a noncovalent complex with either prekallikrein or factor XI, two othe r plasma glycoproteins. The binding domain for factor XI within kinino gen, Pro556-Met613 (58 residues), wholly contains the binding domain f or prekallikrein, Ser565-Lys595 (31 residues), but Trp569-Lys595 (27 r esidues) retains some ability to bind prekallikrein. Complex formation between these proteins is mediated by recognition between complementa ry domains. The 58-residue factor XI peptide domain has now been prepa red following a strategy of condensation of long-chain peptide fragmen ts prepared using orthogonal chemistry protocols. The 58-, 31-, and 27 -residue peptides assume very different structures in aqueous solution as revealed by differential scanning calorimetry, intrinsic fluoresce nce emission, and circular dichroism spectroscopies. Thus, the 31-resi due peptide shows a broad endothermic transition in differential scann ing calorimetry (DSC), but the 58-mer undergoes a well-defined, two-st ate transition (Tm 43-degrees-C; transition enthalpy almost-equal-to 3 0 kcal/mol). The 58- and 27-residue peptides continuously lose structu re with increasing temperature, but the 31-mer retains significant str ucture even at temperatures approaching 90-degrees-C. Lys595 plays a c ritical role in maintaining structure through electrostatic contacts, probably with Asp572 in the N-terminal segment of the 31-residue seque nce. Isothermal ligand titration calorimetry was used to directly asse ss the ability of the 31-, 27-, and 58-residue peptides to bind prekal likrein. The 3 1 -residue peptide binds prekallikrein with 25-fold hig her affinity (K(d) = 1.0 X 10(-6) M) than the 58-residue peptide and w ith 5.4-fold higher affinity than the 27-residue peptide. Hence, the e ssential features of the 31-residue peptide domain required for bindin g prekallikrein are absent in the 58-residue peptide, which is optimiz ed for binding factor XI. The results suggest that a conformational ch ange may occur within kininogen that causes expression of one domain s tructure in preference to the other.