NMR structures reveal how oxidation inactivates thrombomodulin.
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.
Wood
Matthew J
MJ
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0359, USA.
Becvar
L Amaya
LA
Prieto
Judith Helena
JH
Melacini
Giuseppe
G
Komives
Elizabeth A
EA
D000595:Amino Acid Sequence; D004789:Enzyme Activation / Q000235:genetics; D004815:Epidermal Growth Factor / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D011486:Protein C / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism; D018180:Thrombomodulin / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D011486:Protein C; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D008715:Methionine; D013917:Thrombin; D007930:Leucine