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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.
Abstract:
Oxidation of Met 388, one of the three linker residues connecting the fourth and fifth EGF-like domains of thrombomodulin (TM), is deleterious for TM activity. An NMR structure of the smallest active fragment of TM (TMEGF45) and a crystal structure of a larger fragment (TMEGF456) bound to thrombin both show that Met 388 is packed into the fifth domain. Using multidimensional NMR, we have solved the structure of TMEGF45 in which Met 388 is oxidized (TMEGF45ox) and the structure of TMEGF45 in which Met 388 is mutated to Leu (TMEGF45ML). Comparison of the structures shows that the fifth domain has a somewhat different structure depending on the residue at position 388, and several of the thrombin-binding residues are packed into the fifth domain in the oxidized protein while they are exposed and free to interact with thrombin in the native structure and the Met-Leu mutant. This observation is consistent with kinetic measurements showing that the K(m) for TMEGF45ox binding to thrombin is 3.3-fold higher than for the native protein. Most importantly, the connection between the two domains, as indicated by interdomain NOEs, appears to be essential for activity. In the TMEGF45ox structure which has a reduced k(cat) for protein C activation by the thrombin-TMEGF45ox complex, interaction between the two domains is lost. Conversely, a tighter connection is observed between the two domains in TMEGF45ML, which has a higher k(cat) for protein C activation by the thrombin-TMEGF45ML complex.
Insights
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.
