Structure of activated thrombin-activatable fibrinolysis inhibitor, a molecular link between coagulation and fibrinolysis.
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a metallocarboxypeptidase (MCP) that links blood coagulation and fibrinolysis. TAFI hampers fibrin-clot lysis and is a pharmacological target for the treatment of thrombotic conditions. TAFI is transformed through removal of its prodomain by thrombin-thrombomodulin into TAFIa, which is intrinsically unstable and has a short half-life in vivo. Here we show that purified bovine TAFI activated in the presence of a proteinaceous inhibitor renders a stable enzyme-inhibitor complex. Its crystal structure reveals that TAFIa conforms to the alpha/beta-hydrolase fold of MCPs and displays two unique flexible loops on the molecular surface, accounting for structural instability and susceptibility to proteolysis. In addition, point mutations reported to enhance protein stability in vivo are mainly located in the first loop and in another surface region, which is a potential heparin-binding site. The protein inhibitor contacts both the TAFIa active site and an exosite, thus contributing to high inhibitory efficiency.
Sanglas
Laura
L
Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular (Facultat de Ciències), Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.
Valnickova
Zuzana
Z
Center for Insoluble Protein Structures at the Department of Molecular Biology, Science Park, University of Århus, Gustav Wieds Vej 10c, DK-8000 Århus C, Denmark.
Arolas
Joan L
JL
Department of Structural Biology at the Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, c/Balidiri Reixach, 10-12 and 15-21, E-08028, Spain.
Pallarés
Irantzu
I
Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular (Facultat de Ciències), Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.
Guevara
Tibisay
T
Department of Structural Biology at the Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, c/Balidiri Reixach, 10-12 and 15-21, E-08028, Spain.
Solà
Maria
M
Department of Structural Biology at the Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, c/Balidiri Reixach, 10-12 and 15-21, E-08028, Spain.
Kristensen
Torsten
T
Center for Insoluble Protein Structures at the Department of Molecular Biology, Science Park, University of Århus, Gustav Wieds Vej 10c, DK-8000 Århus C, Denmark.
Enghild
Jan J
JJ
Center for Insoluble Protein Structures at the Department of Molecular Biology, Science Park, University of Århus, Gustav Wieds Vej 10c, DK-8000 Århus C, Denmark.
Aviles
Francesc X
FX
Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular (Facultat de Ciències), Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain. Electronic address: francescxavier.aviles@uab.es.
Gomis-Rüth
F Xavier
FX
Department of Structural Biology at the Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, c/Balidiri Reixach, 10-12 and 15-21, E-08028, Spain. Electronic address: xgrcri@ibmb.csic.es.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D001777:Blood Coagulation; D025901:Carboxypeptidase B2 / Q000737:chemistry* / Q000302:isolation & purification; D002417:Cattle; D018360:Crystallography, X-Ray; D005342:Fibrinolysis; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013816:Thermodynamics
D006493:Heparin; D025901:Carboxypeptidase B2