Evidence for multiple conformational changes in the active center of thrombin induced by complex formation with thrombomodulin: an analysis employing nitroxide spin-labels.
Thrombomodulin (TM) is an endothelial cell surface protein that binds thrombin to form a reversible complex with altered enzyme specificity. The complex rapidly converts protein C to the anticoagulant enzyme activated protein C and has decreased fibrinogen clotting activity. To investigate whether formation of this complex elicits conformational changes in the active center of thrombin, we employed the following fluorosulfonyl spin-label inhibitors: N-(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-m-(fluorosulfonyl)benzamide (m-V); O-(2,2,6,6-tetramethyl-1-oxy-4-piperidinyl) N-[m-(fluorosulfonyl)phenyl]carbamate (m-VI); N-[4-(fluorosulfonyl)phenyl]-2,2,5,5-tetramethyl-1-oxy-3-pyrroline -3-carboxamide (p-I); N-(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-p-(fluorosulfonyl)benzamide (p-V). To compare the spectra of the free thrombin with those of the complex, the viscosity of the solution was adjusted with sucrose to give similar tumbling rates (isokylindric spectra) or the macromolecular rotational contribution to the spectra was essentially eliminated with saturated sucrose. Both a buffer-soluble proteolytic derivative of TM and the intact molecule elicited changes in the electron spin resonance signals of many of the labeled thrombins employed. Two of the labels, p-I and p-V, had previously been shown to exhibit decreased mobility when indole derivatives were bound to thrombin. When TM complexes with thrombin, the mobility of the p-I label increases while the mobility of the p-V label decreases.(ABSTRACT TRUNCATED AT 250 WORDS)
Musci
G
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Department of Chemistry, Ohio State University, Columbus 43210.
Berliner
L J
LJ
Esmon
C T
CT
D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D011487:Protein Conformation; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013113:Spin Labels; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism*
D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013113:Spin Labels; D013917:Thrombin