title: Conformational analysis of synthetic peptides encompassing the factor XI and prekallikrein overlapping binding domains of high molecular weight kininogen.
abstract: High molecular weight kininogen, a plasma glycoprotein, circulates as a noncovalent complex with either prekallikrein or factor XI, two other plasma glycoproteins. The binding domain for factor XI within kininogen, Pro556-Met613 (58 residues), wholly contains the binding domain for prekallikrein, Ser565-Lys595 (31 residues), but Trp569-Lys595 (27 residues) retains some ability to bind prekallikrein. Complex formation between these proteins is mediated by recognition between complementary domains. The 58-residue factor XI peptide domain has now been prepared following a strategy of condensation of long-chain peptide fragments 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 fluorescence emission, and circular dichroism spectroscopies. Thus, the 31-residue peptide shows a broad endothermic transition in differential scanning calorimetry (DSC), but the 58-mer undergoes a well-defined, two-state transition (Tm 43 degrees C; transition enthalpy approximately 30 kcal/mol). The 58- and 27-residue peptides continuously lose structure with increasing temperature, but the 31-mer retains significant structure even at temperatures approaching 90 degrees C. Lys595 plays a critical role in maintaining structure through electrostatic contacts, probably with Asp572 in the N-terminal segment of the 31-residue sequence. Isothermal ligand titration calorimetry was used to directly assess the ability of the 31-, 27-, and 58-residue peptides to bind prekallikrein. The 31-residue peptide binds prekallikrein with 25-fold higher affinity (Kd = 1.0 x 10(-6) M) than the 58-residue peptide and with 5.4-fold higher affinity than the 27-residue peptide. Hence, the essential features of the 31-residue peptide domain required for binding prekallikrein are absent in the 58-residue peptide, which is optimized for binding factor XI. The results suggest that a conformational change may occur within kininogen that causes expression of one domain structure in preference to the other.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298-0614.
authors.forename: J L
authors.initials: JL
authors.lastname: You
authors.forename: J D
authors.initials: JD
authors.lastname: Page
authors.forename: J N
authors.initials: JN
authors.lastname: Scarsdale
authors.forename: R W
authors.initials: RW
authors.lastname: Colman
authors.forename: R B
authors.initials: RB
authors.lastname: Harris
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002152:Calorimetry, Differential Scanning; D005172:Factor XI / Q000737:chemistry*; D007704:Kininogens / Q000737:chemistry*; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry*; D011288:Prekallikrein / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D017186:Titrimetry
chemical_list: D007704:Kininogens; D008024:Ligands; D010455:Peptides; D005172:Factor XI; D011288:Prekallikrein