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Related Experiment Videos

Allosteric changes in solvent accessibility observed in thrombin upon active site occupation.

Carrie Hughes Croy1, Julia R Koeppe, Simon Bergqvist

  • 1Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0378, USA.

Biochemistry
|May 5, 2004
PubMed
Summary

Comparing thrombin

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Dynamics

Background:

  • Thrombin's active site structure and function are crucial for hemostasis.
  • Previous studies on thrombin-thrombomodulin complexes showed conflicting results regarding active site changes.
  • Understanding substrate-induced conformational changes in thrombin is key to its regulation.

Purpose of the Study:

  • To compare solvent accessibility of thrombin in substrate-free and substrate-bound states.
  • To investigate allosteric pathways connecting the active site to distal regions.
  • To reconcile conflicting data from crystal structures and hydrogen/deuterium exchange studies.

Main Methods:

  • Amide hydrogen/deuterium (H/2H) exchange was employed to assess solvent accessibility.

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  • The optimized inhibitor peptide dPhe-Pro-Arg chloromethyl ketone (PPACK) simulated substrate binding.
  • Analysis focused on changes in active site loops and distal regions upon PPACK binding.

    • Main Results:

    • Two active site loops (residues 214-222 and 126-132) showed decreased solvent accessibility with PPACK.
    • Two regions outside the active site, including anion binding exosite 1, gained solvent protection.
    • A beta-strand loop containing Trp141, linked to exosite 1, also showed protection.

    Conclusions:

    • Substrate binding to thrombin induces conformational changes in both active site and distal regions.
    • Allosteric changes are transmitted from the active site to anion binding exosite 1 via specific pathways.
    • These findings provide insights into thrombin's allosteric regulation and substrate recognition.