Activation of protein C by the thrombin-thrombomodulin complex: cooperative roles of Arg-35 of thrombin and Arg-67 of protein C.
The binding of Ca(2+) to the 70-80 loop of protein C inhibits protein C activation by thrombin in the absence of thrombomodulin (TM), but the metal ion is required for activation in the presence of TM. Structural data suggests that the 70-80 loop is located between two antiparallel beta strands comprised of residues 64-69 and 81-91 on the protease domain of protein C. To test the hypothesis that a salt-bridge/hydrogen bond interaction between Arg-67 of the former strand and Asp-82 of the latter strand modulates the unique Ca(2+)-binding properties of protein C, we engineered a disulfide bond between the two strands by substituting both Arg-67 and Asp-82 with Cys residues. The activation of this mutant was enhanced 40- to 50-fold independent of TM and Ca(2+). Furthermore, the Arg-67 to Ala mutant of protein C was activated in the absence of TM by the Arg-35 to Glu mutant of thrombin with the same efficiency as wild-type protein C by wild-type thrombin-TM complex. These results suggest that TM functions by alleviating the Ca(2+)-dependent inhibitory interactions of Arg-67 of protein C and Arg-35 of thrombin.
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Manithody
Chandrashekhara
C
Rezaie
Alireza R
AR
D000409:Alanine / Q000737:chemistry; D000991:Antithrombins / Q000737:chemistry; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry* / Q000502:physiology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D018179:Receptors, Thrombin / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry* / Q000502:physiology; D018180:Thrombomodulin / Q000737:chemistry* / Q000502:physiology; D013997:Time Factors
D000991:Antithrombins; D011486:Protein C; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D018180:Thrombomodulin; D001120:Arginine; D004492:Edetic Acid; D013917:Thrombin; D000409:Alanine; D002118:Calcium