The fourth epidermal growth factor-like domain of thrombomodulin interacts with the basic exosite of protein C.
Thrombomodulin (TM) functions as a cofactor to enhance the rate of protein C activation by thrombin approximately 1000-fold. The molecular mechanism by which TM improves the catalytic efficiency of thrombin toward protein C is not known. Molecular modeling of the protein C activation based on the crystal structure of thrombin in complex with the epidermal growth factor-like domains 4, 5, and 6 of TM (TM456) predicts that the binding of TM56 to exosite 1 of thrombin positions TM4 so that a negatively charged region on this domain juxtaposes a positively charged region of protein C. It has been hypothesized that electrostatic interactions between these oppositely charged residues of TM4 and protein C facilitate a proper docking of the substrate into the catalytic pocket of thrombin. To test this hypothesis, we have constructed several mutants of TM456 and protein C in which charges of the putative interacting residues on both TM4 (Asp/Glu) and protein C (Lys/Arg) have been reversed. Results of TM-dependent protein C activation studies by such a compensatory mutagenesis approach support the molecular model that TM4 interacts with the basic exosite of protein C.
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104, USA.
Rezaie
Alireza R
AR
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011486:Protein C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D018180:Thrombomodulin / Q000737:chemistry* / Q000378:metabolism
D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D013917:Thrombin; D002118:Calcium