Fine mapping of the sequences in domain 5 of high molecular weight kininogen (HK) interacting with heparin and zinc

J Thromb Haemost. 2003 Aug;1(8):1791-8. doi: 10.1046/j.1538-7836.2003.00291.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antithrombins / chemistry
  • Biotinylation
  • Dose-Response Relationship, Drug
  • Edetic Acid / pharmacology
  • Gene Deletion
  • Glutathione Transferase / metabolism
  • Heparin / chemistry*
  • Histidine / chemistry
  • Humans
  • Kinetics
  • Kininogen, High-Molecular-Weight / chemistry*
  • Kininogen, High-Molecular-Weight / genetics
  • Lysine / chemistry
  • Models, Molecular
  • Molecular Sequence Data
  • Peptides / chemistry
  • Protein Binding
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Surface Plasmon Resonance
  • Thrombin / chemistry
  • Time Factors
  • Zinc / chemistry*

Substances

  • Antithrombins
  • Kininogen, High-Molecular-Weight
  • Peptides
  • Histidine
  • Heparin
  • Edetic Acid
  • Glutathione Transferase
  • Thrombin
  • Zinc
  • Lysine