Mutagenesis studies toward understanding allostery in thrombin.
Mutagenesis studies toward understanding allostery in thrombin.
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DOI：
10.1021/bi900921t
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发表时间：
2009-09-01
期刊：
影响因子：
2.9
通讯作者：
Rezaie, Alireza R.
中科院分区：
文献类型：
--
作者：
Qureshi, Shabir H.;Yang, Likui;Manithody, Chandrashekhara;Iakhiaev, Alexei V.;Rezaie, Alireza R.
血栓调节蛋白（TM）与exosite-1的结合和Na+与225环的结合变构调节凝血酶的催化活性和底物特异性。为了确定这两个辅因子结合环的构象是否相互连接并与活性位点连接，我们合理地设计了两个凝血酶突变体，其中70 - 80环的外切位点-1或225环的Na+结合位点通过工程二硫键稳定。这可以通过用Cys残基替换第一突变体中的两个残基Arg-67和Ile-82以及第二突变体中的两个残基Glu-217和Lys-224来实现。这些突变体在哺乳动物细胞中表达为单体分子，纯化至均一性，并通过动力学和直接结合方法表征其结合TM和Na+的能力。Cys-67/Cys-82突变体不与TM结合，并表现出正常的酰胺分解活性，然而，Cys-217/Cys-224的活性显著受损，尽管TM与该突变体以> 20倍升高的KD相互作用以部分恢复其活性。在TM存在下，两种突变体与Na+的相互作用均表现出高10.2 - 3倍的KD，并且突变体均不凝结纤维蛋白原或活化蛋白C。两种突变体与肝素的相互作用具有正常的亲和力。这些结果表明，虽然凝血酶的exosite-2是一个独立的辅因子结合位点，但Na+结合和exosite-1都是能量连接的。用荧光素标记的凝血酶Cys-195突变体进一步研究表明，凝血酶的催化残基受Na+的调节，但TM对该残基的构象没有影响。
The binding of thrombomodulin (TM) to exosite-1 and the binding of Na+ to 225-loop allosterically modulate the catalytic activity and substrate specificity of thrombin. To determine whether the conformation of these two cofactor-binding loops are energetically linked to each other and to the active-site, we rationally designed two thrombin mutants in which either the 70-80 loop of exosite-1 or the 225-loop of the Na+-binding site was stabilized by an engineered disulfide bond. This was possible by replacing two residues, Arg-67 and Ile-82, in the first mutant and two residues, Glu-217 and Lys-224, in the second mutant with Cys residues. These mutants were expressed in mammalian cells as monomeric molecules, purified to homogeneity and characterized with respect to their ability to bind TM and Na+ by kinetic and direct binding approaches. The Cys-67/Cys-82 mutant did not bind TM and exhibited a normal amidolytic activity, however, the activity of Cys-217/Cys-224 was dramatically impaired, though TM interacted with this mutant with >20-fold elevated KD to partially restore its activity. Both mutants exhibited ∼2-3-fold higher KD for interaction with Na+ and neither mutant clotted fibrinogen or activated protein C in the presence of TM. Both mutants interacted with heparin with a normal affinity. These results suggest that, while exosite-2 of thrombin is an independent cofactor binding-site, both the Na+-binding and exosite-1 are energetically linked. Further studies with the fluorescein labeled Cys-195 mutant of thrombin revealed that the catalytic residue of thrombin is modulated by Na+, but TM has no effect on the conformation of this residue.
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影响因子：
2.9
作者：
DAVIE, EW;FUJIKAWA, K;KISIEL, W
通讯作者：
KISIEL, W
影响因子：
2.9
作者：
ARNI, RK;PADMANABHAN, K;TULINSKY, A
通讯作者：
TULINSKY, A
影响因子：
4.8
作者：
Anderson, PJ;Nesset, A;Bock, PE
通讯作者：
Bock, PE
影响因子：
4.8
作者：
Bush-Pelc, Leslie A.;Marino, Francesca;Di Cera, Enrico
通讯作者：
Di Cera, Enrico
影响因子：
4.8
作者：
He, XH;Rezaie, AR
通讯作者：
Rezaie, AR