Fine mapping of the sequences in domain 5 of high molecular weight kininogen (HK) interacting with heparin and zinc.
We previously localized the heparin binding region on high molecular weight kininogen to domain 5 (D5) by quantifying the binding using surface plasmon resonance of D5 fused at its N-terminal to glutathione-S-transferase. We further examined GST-(H475-S626) which at 100 nm was previously shown to be ineffective in reversing the heparin acceleration of antithrombin inhibition of thrombin. However, we now show that at a concentration of 400 nm, complete reversal of accelerated inhibition occurred. To characterize the interacting sequences on D5, four peptides representing surface loops of a molecular model were synthesized. Peptides H475-H485 and G440-G455, rich in histidine and low in lysine, showed weak or no detectable binding in the absence of Zn++, but tighter binding in the presence of Zn++. H483-K497 containing three histidines and six lysines showed tight binding without Zn++, and increased in avidity with Zn++. In contrast, G486-K502, low in histidine and high in lysine, showed tight binding (KD = 0.8 microm) in the absence and presence of Zn++. Both H483-K497 and G486-K502 were effective in neutralizing the accelerated inhibition by heparin of thrombin by antithrombin in the absence of Zn++. Therefore, a set of lysine residues in the sequence of K487-K502 is responsible for Zn++-independent binding of heparin. Further, a group of histidine residues in sequence range of H475-H485 contributes to Zn++-dependent binding of heparin to HK-D5.
Pixley
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Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA. robin.pixley@temple.edu
Lin
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Y
Isordia-Salas
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I
Colman
R W
RW
D000595:Amino Acid Sequence; D000991:Antithrombins / Q000737:chemistry; D019921:Biotinylation; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006493:Heparin / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D019679:Kininogen, High-Molecular-Weight / Q000737:chemistry* / Q000235:genetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors; D015032:Zinc / Q000737:chemistry*
D000991:Antithrombins; D019679:Kininogen, High-Molecular-Weight; D010455:Peptides; D006639:Histidine; D006493:Heparin; D004492:Edetic Acid; D005982:Glutathione Transferase; D013917:Thrombin; D015032:Zinc; D008239:Lysine