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Related Experiment Videos

Atomic structures of two nitroxide spin labels complexed with human thrombin: comparison with solution studies.

V L Nienaber1, L J Berliner

  • 1Department of Chemical and Physical Sciences, The DuPont Merck Pharmaceutical Company, Experimental Station, Wilmington, Delaware 19880, USA.

Journal of Protein Chemistry
|August 17, 2000
PubMed
Summary

Crystal structures reveal how spin labels para-V and meta-V bind to thrombin. These findings map thrombin's active site, complementing solution studies.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Thrombin is a key enzyme in blood coagulation.
  • Electron spin resonance (ESR) studies previously mapped thrombin's active site using spin labels.
  • Understanding thrombin's active site is crucial for developing anticoagulants.

Purpose of the Study:

  • To determine the crystal structures of thrombin complexed with para-V and meta-V spin labels.
  • To elucidate the binding interactions of these spin labels within thrombin's active site.
  • To integrate structural data with previous ESR findings for a comprehensive active site map.

Main Methods:

  • X-ray crystallography was used to obtain high-resolution crystal structures (2.0 and 3.0 Å).
  • Complexes of thrombin with para-V (4-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-(fluorosulfonyl) benzamidine) and meta-V (3-(2,2,5,5-tetramethyl-pyrrolidine1-oxyl)-m-(fluorosulfonyl) benzamidine) were crystallized.

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  • Structural analysis focused on the binding locations and interactions of the spin labels.

    • Main Results:

    • The crystal structure of thrombin complexed with para-V was determined at 2.0 Å resolution.
    • The crystal structure of thrombin complexed with meta-V was determined at 3.0 Å resolution.
    • Para-V binds within the substrate binding cleft, while meta-V binds at the primary specificity pocket and a site interacting with the gamma-cleavage loop.

    Conclusions:

    • The crystal structures provide precise atomic details of spin label binding to thrombin.
    • These findings validate and refine the low-resolution topography map obtained from ESR studies.
    • The study highlights the complementary nature of solid-state (crystallography) and solution (ESR) biophysical techniques for studying enzyme active sites.