Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli:: Comparison with functionally related proteins
Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli:: Comparison with functionally related proteins
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DOI：
10.1006/jmbi.2001.4767
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发表时间：
2001-07-06
影响因子：
5.6
通讯作者：
Nilsson, L
中科院分区：
文献类型：
--
作者：
Foloppe, N;Sagemark, J;Nilsson, L
活性位点半胱氨酸残基的化学对于硫氧还蛋白超家族的硫醇-二硫键氧化还原酶的活性至关重要。在这些反应中，需要亲核硫醇盐，但相关的pK（a），值在超家族中差异很大，从DsbA中的小于4到Trx中的大于7。然而，稳定这种硫醇盐的因素尚未明确确定。谷氧还蛋白（Grxs）是该超家族的成员，在其活性位点含有Cys-Pro-Tyr-Cys基序。在还原的Grxs中，N-末端活性位点亲核半胱氨酸残基的pK（a）显著降低，并且预期相应硫醇盐的稳定性会影响这些酶的氧化还原电位。在这里，我们使用长的分子动力学（MD）模拟，pK（a）计算和实验研究的组合，以获得从大肠杆菌Grx 3的减少的活性位点的结构和动力学，并调查稳定硫醇的因素。几个不同的MD模拟收敛到一个共识的构象的活性位点半胱氨酸残基（Cys 11和Cys 14），一些局部构象变化后。通过测量NMR标量耦合常数和测定所选残基的pK（a）值，对模型的关键特征进行了实验测试。在MD模拟过程中计算了Grx 3活性位点残基的pK（a）值，并支持基础结构模型。Grx 3的结构与pK（a）计算相结合表明，Grx 3中N-末端活性位点半胱氨酸残基的pK（a）介于DsbA和Trx中对应物的pK（a）之间。pK（a）值与实验最吻合，在低（
The chemistry of active-site cysteine residues is central to the activity of thiol-disulfide oxidoreductases of the thioredoxin superfamily. In these reactions, a nucleophilic thiolate is required, but the associated pK(a), values differ vastly in the superfamily, from less than 4 in DsbA to greater than 7 in Trx. The factors that stabilize this thiolate are, however, not clearly established. The glutaredoxins (Grxs), which are members of this superfamily, contain a Cys-Pro-Tyr-Cys motif in their active site. In reduced Grxs, the pK(a) of the N-terminal active-site nucleophilic cysteine residue is lowered significantly, and the stabilization of the corresponding thiolate is expected to influence the redox potential of these enzymes. Here, we use a combination of long molecular dynamics (MD) simulations, pK(a) calculations, and experimental investigations to derive the structure and dynamics of the reduced active site from Escherichia coli Grx3, and investigate the factors that stabilize the thiolate. Several different MD simulations converged toward a consensus conformation for the active-site cysteine residues (Cys11 and Cys14), after a number of local conformational changes. Key features of the model were tested experimentally by measurement of NMR scalar coupling constants, and determination of pK(a) values of selected residues. The pK(a) values of the Grx3 active-site residues were calculated during the MD simulations, and support the underlying structural model. The structure of Grx3, in combination with the pK(a) calculations, indicate that the pK(a) of the N-terminal active-site cysteine residue in Grx3 is intermediate between that of its counterpart in DsbA and Trx. The pK(a) values in best agreement with experiment are obtained with a low (