SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase.
SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase.
复制标题
SUFE D74R替代改变了主动位点环动力学，以进一步增强SUFE与Sufs半胱氨酸脱硫酶的相互作用。
DOI：
10.1021/acs.biochem.5b00663
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发表时间：
2015-08-11
期刊：
影响因子：
2.9
通讯作者：
Outten FW
中科院分区：
文献类型：
--
作者：
Dai Y;Kim D;Dong G;Busenlehner LS;Frantom PA;Outten FW
许多必需的金属蛋白需要铁 - 硫（Fe - S）簇辅因子来发挥其功能。在大多数生物体中，由L - 半胱氨酸形成过硫化物是铁 - 硫簇生物合成的关键步骤。在大肠杆菌中，SufS半胱氨酸脱硫酶通过一种依赖磷酸吡哆醛（PLP）的乒乓反应从L - 半胱氨酸动员过硫化物。SufS需要SufE伴侣蛋白将过硫化物转移到SufB铁 - 硫簇支架上。没有SufE，SufS酶无法高效周转，并保持在与过硫化物结合的状态。协同的蛋白质 - 蛋白质相互作用介导硫从SufS转移到SufE。多项研究表明，在硫从SufS转移过程中，SufE必须经历构象变化以伸展其活性位点的半胱氨酸环。为了验证这一假定的模型，我们将SufE的天冬氨酸74突变为精氨酸（D74R）以增加SufE半胱氨酸51环的动态性。对SufE D74R进行的酰胺氢/氘交换质谱（HDX - MS）分析显示，包含用于从SufS接受过硫化物的活性位点半胱氨酸51的环的溶剂可及性和动态性增加。我们的结果表明，突变蛋白对SufS的结合亲和力比野生型SufE更强。此外，与野生型SufE不同，SufE D74R仍然能够增强SufS脱硫酶活性，并且在较高的SufE D74R浓度下未显示饱和。这些结果表明，动态变化可能使SufE转变为一种与SufS相互作用更强的硫接受态。
Many essential metalloproteins require iron–sulfur (Fe–S) cluster cofactors for their function. In vivo persulfide formation from L-cysteine is a key step in the biogenesis of Fe–S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from L-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe–S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein–protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS.