Contiguous binding and inhibitory sites on kininogens required for the inhibition of platelet calpain.
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.
Bradford
H N
HN
Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Jameson
B A
BA
Adam
A A
AA
Wassell
R P
RP
Colman
R W
RW
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
D007704:Kininogens; D002154:Calpain