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Related Experiment Videos

Evidence for common structural changes in thrombin induced by active-site or exosite binding

M A Parry1, S R Stone, J Hofsteenge

  • 1Friedrich Miescher-Institut, Basel, Switzerland.

The Biochemical Journal
|March 15, 1993
PubMed
Summary

Ligand binding to thrombin’s active site or exosite induces structural changes in the gamma-loop, reducing its susceptibility to proteolysis. This allosteric interaction suggests a novel mechanism for thrombin regulation.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein structure and function

Background:

  • The gamma-loop of thrombin is a flexible, surface-accessible region involved in enzyme interactions.
  • Understanding structural changes in thrombin is crucial for comprehending its regulatory mechanisms.

Purpose of the Study:

  • To investigate structural alterations in thrombin's gamma-loop induced by site-specific ligands.
  • To elucidate the allosteric communication between thrombin's active site, exosites, and the gamma-loop.

Main Methods:

  • Limited proteolysis assays to assess gamma-loop susceptibility to cleavage by elastase and chymotrypsin.
  • Intrinsic fluorescence measurements to detect conformational changes in thrombin.
  • Utilizing site-specific ligands targeting the anion-binding exosite and active site.

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Main Results:

  • Binding of hirudin peptide to the anion-binding exosite reduced gamma-loop proteolysis susceptibility by 6-fold.
  • Active site inhibitors, like D-Phe-Pro-Arg-CH2Cl, provided greater protection, reducing chymotrypsin cleavage by 95-fold.
  • Both exosite and active site ligand binding induced a common intrinsic fluorescence change, indicating an allosteric effect.

Conclusions:

  • A novel allosteric interaction couples thrombin's active site to the gamma-loop, independent of direct physical contact.
  • This allosteric mechanism may explain how thrombomodulin modulates thrombin's enzymatic activity.
  • Structural plasticity of the gamma-loop is influenced by ligand binding at distant sites on the thrombin molecule.