title: Contiguous binding and inhibitory sites on kininogens required for the inhibition of platelet calpain.
abstract: Both high molecular weight kininogen (HK) and low molecular weight kininogens (LK) are potent tight binding inhibitors of platelet calpain (Ki = 2 nM), but the molecular basis for the inhibitory function is not well delineated. The amino acid sequences of the calpain inhibitory domain 2 from human and rat HK were compared for homology with the noninhibitory domains from human and rat domain 3 and from domain 2 of rat T-kininogen, and two areas of nonconserved differences were detected. Computer three-dimensional models were constructed on a template built using the x-ray crystallographic data for cystatin, an evolutionary precursor of HK. Two nonconserved regions in the calpain inhibitory domains flank the highly conserved motif QVVAG to form a continuous surface for interaction with cysteine proteases. Three peptide sequences, components of the modeled surface, were chosen for synthesis from HK D-2: VHPISTQSPDLE (peptide 146-156, NH2-terminal), CTDNAYIDIQLRIASFSQNC (peptide 229-248, COOH-terminal), and CQRQVVAGLNFRIC (185-189, central) containing QVVAG. This last peptide differs from the natural sequence by substitutions of A185C and T195C. Peptides 185-198 and 229-248 were folded by air oxidation of their cysteine residues and then tested for their ability to inhibit calpain and papain. The folded peptide 229-248 inhibited calpain with an IC50 35 microM and unfolding reduced this effect. The folded peptide 185-198 did not inhibit calpain, but when preincubated with calpain, could block the inhibition by HK indicating a probable enzyme binding site. Peptide 146-157 did not inhibit calpain but could inhibit papain with an IC50 of 20 microM. We have thus defined separate binding and inhibitory sequences on HK which form a contiguous surface for thiol protease interactions.
authors.affiliation: Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
authors.forename: H N
authors.initials: HN
authors.lastname: Bradford
authors.forename: B A
authors.initials: BA
authors.lastname: Jameson
authors.forename: A A
authors.initials: AA
authors.lastname: Adam
authors.forename: R P
authors.initials: RP
authors.lastname: Wassell
authors.forename: R W
authors.initials: RW
authors.lastname: Colman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors*; D003196:Computer Graphics; D006801:Humans; D007704:Kininogens / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid
chemical_list: D007704:Kininogens; D002154:Calpain