Thrombomodulin tightens the thrombin active site loops to promote protein C activation.
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. Crystallographic investigations of the complex between thrombin and TMEGF456 did not show any changes in the thrombin active site. Therefore, research has focused recently on how TM may provide a docking site for the protein C substrate. Previous work, however, showed that when the thrombin active site was occupied with substrate analogues labeled with fluorophores, the fluorophores responded differently to active (TMEGF1-6) versus inactive (TMEGF56) fragments of TM. To investigate this further, we have carried out amide H/(2)H exchange experiments on thrombin in the presence of active (TMEGF45) and inactive (TMEGF56) fragments of TM. Both on-exchange and off-exchange experiments show changes in the thrombin active site loops, some of which are observed only when the active TM fragment is bound. These results are consistent with the previously observed fluorescence changes and point to a mechanism by which TM changes the thrombin substrate specificity in favor of protein C cleavage.
Koeppe
Julia R
JR
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0378, USA.
Seitova
Almagoul
A
Mather
Timothy
T
Komives
Elizabeth A
EA
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D011486:Protein C / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D018180:Thrombomodulin / Q000737:chemistry* / Q000378:metabolism
D000577:Amides; D011486:Protein C; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D013917:Thrombin