Dynamics of the fragment of thrombomodulin containing the fourth and fifth epidermal growth factor-like domains correlate with function.
Thrombomodulin (TM) forms a 1:1 complex with thrombin. Whereas thrombin alone cleaves fibrinogen to make the fibrin clot, the thrombin-TM complex cleaves protein C to initiate the anticoagulant pathway. The fourth and fifth EGF-like domains of TM together form the minimal fragment with anticoagulant cofactor activity. A short linker connects the fourth and fifth EGF-like domains of TM, and Met 388 in the middle of the linker interacts with both domains. Several different structures of TMEGF45 variants are now available, and these show that mutation of Met 388 alters the structure of the fifth domain, as well as the connectivity of the two domains. To probe this phenomenon more thoroughly, NMR backbone dynamics experiments have been carried out on the individual fourth and fifth domains as well as on the wild type, the Met 388 Leu mutant, and the variant in which Met 388 is oxidized. The results presented here show that changes at Met 388 cause significant changes in backbone dynamics in both the fourth and fifth EGF-like domains of TM. Backbone dynamics within the small loop of the fourth domain Tyr 358 correlate with anticoagulant cofactor activity. Backbone dynamics of the thrombin-binding residues Tyr 413 and Ile 414 are inversely correlated with thrombin binding. The preordering of the backbone of Tyr 413 and Ile 414 only occurs in the two-domain fragments, revealing a role for the fourth domain in thrombin binding as well as in anticoagulant cofactor activity.
Prieto
Judith H
JH
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0378, USA.
Sampoli Benitez
Benedetta A
BA
Melacini
Giuseppe
G
Johnson
David A
DA
Wood
Matthew J
MJ
Komives
Elizabeth A
EA
D019943:Amino Acid Substitution / Q000235:genetics; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000502:physiology; D005454:Fluorescence Polarization; D007930:Leucine / Q000235:genetics; D008715:Methionine / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D018180:Thrombomodulin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
D010446:Peptide Fragments; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D008715:Methionine; D007930:Leucine