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NMR structures reveal how oxidation inactivates thrombomodulin.
Matthew J Wood1, L Amaya Becvar, Judith Helena Prieto
1Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0359, USA.
Biochemistry
|October 15, 2003
Summary
Oxidation of methionine 388 in thrombomodulin (TM) impairs its activity by altering domain structure and reducing thrombin binding. Maintaining the connection between TM domains is crucial for protein C activation efficacy.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Medicine
Background:
- Thrombomodulin (TM) is a crucial regulator of blood coagulation.
- Met 388, a linker residue in TM, is vital for its activity.
- Oxidation of Met 388 negatively impacts TM function.
Purpose of the Study:
- To elucidate the structural consequences of Met 388 oxidation in TM.
- To understand the role of Met 388 in TM-thrombin interactions.
- To investigate the impact of Met 388 modification on protein C activation.
Main Methods:
- Multidimensional NMR spectroscopy was used to determine the structures of TM fragments.
- Comparison of native, oxidized (TMEGF45ox), and Met-to-Leu mutated (TMEGF45ML) TM structures.
- Kinetic measurements (K(m) and k(cat)) were performed to assess TM activity.
Main Results:
- Oxidation of Met 388 alters the structure of the fifth EGF-like domain.
- Oxidized TM shows reduced binding affinity (higher K(m)) to thrombin.
- Loss of interdomain connection in TMEGF45ox correlates with reduced catalytic efficiency (k(cat)).
Conclusions:
- Met 388 oxidation disrupts the structural integrity and function of thrombomodulin.
- The interdomain connection within TM is essential for efficient protein C activation.
- Met-to-Leu mutation preserves domain interaction and enhances catalytic activity.