Insights into the molecular inactivation mechanism of human activated thrombin-activatable fibrinolysis inhibitor

被引:14
作者
Sanglas, L. [2 ,3 ]
Arolas, J. L. [1 ]
Valnickova, Z. [4 ,5 ]
Aviles, F. X. [2 ,3 ]
Enghild, J. J. [4 ,5 ]
Gomis-Ruth, F. X. [1 ]
机构
[1] CSIC, Proteolysis Lab, Dept Biol Struct, Mol Biol Inst Barcelona, E-08028 Barcelona, Spain
[2] Univ Autonoma Barcelona, Inst Biotecnol & Biomed, Bellaterra, Spain
[3] Univ Autonoma Barcelona, Fac Ciencies, Dept Bioquim & Biol Mol, Bellaterra, Spain
[4] Aarhus Univ, Ctr Insoluble Prot Struct inSPIN, Aarhus, Denmark
[5] Aarhus Univ, Interdisciplinary Nanosci Ctr iNANO, Dept Mol Biol, Aarhus, Denmark
基金
新加坡国家研究基金会;
关键词
blood coagulation; metallopeptidase; TAFI; zymogen activation; CARBOXYPEPTIDASE INHIBITOR; PLASMA CARBOXYPEPTIDASE; PROCARBOXYPEPTIDASE-B; PROTEOLYTIC CLEAVAGE; THERMAL-STABILITY; STRUCTURAL BASIS; U TAFI; ENZYME; METALLOCARBOXYPEPTIDASES; PROTEIN;
D O I
10.1111/j.1538-7836.2010.03740.x
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a validated target for thrombotic diseases. TAFI is converted in vivo to activated TAFI (TAFIa) by removal of its pro-domain. Whereas TAFI is stable and persists in the circulation, possibly in complex with plasminogen, TAFIa is unstable and poorly soluble, with a half-life of minutes.Objectives: In order to study the molecular determinants of this instability, we studied the influence of protein inhibitors on human TAFIa. Results: We found that protein inhibitors significantly reduced the instability and insolubility of TAFIa. In addition, we solved the 2.5-A resolution crystal structure of human TAFIa in complex with a potent protein inhibitor, tick-derived carboxypeptidase inhibitor, which gives rise to a stable and soluble TAFIa species. The structure revealed a significant reduction in the flexibility of dynamic segments when compared with the structures of bovine and human TAFI. We also identified two latent hotspots, loop L beta 2 beta 3 and segment alpha 5-L alpha 5 beta 7-beta 7, where conformational destabilization may begin. These hotspots are also present in TAFI, but the pro-domain may provide sufficient stabilization and solubility to guarantee protein persistence in vivo. When the pro-domain is removed, the free TAFIa moiety becomes unstable, its activity is suppressed, and the molecule becomes insoluble. Conclusions: The present study corroborates the function of protein inhibitors in stabilizing human TAFIa and it provides a rigid and high-resolution mold for the design of small molecule inhibitors of this enzyme, thus paving the way for novel therapy for thrombotic disorders.
引用
收藏
页码:1056 / 1065
页数:10
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