Changes in Protein Dynamics in Escherichia coli SufS Reveal a Possible Conserved Regulatory Mechanism in Type II Cysteine Desulfurase Systems.
Changes in Protein Dynamics in Escherichia coli SufS Reveal a Possible Conserved Regulatory Mechanism in Type II Cysteine Desulfurase Systems.
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DOI：
10.1021/acs.biochem.7b01275
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发表时间：
2018-09-04
期刊：
影响因子：
2.9
通讯作者：
Frantom PA
中科院分区：
文献类型：
--
作者：
Kim D;Singh H;Dai Y;Dong G;Busenlehner LS;Outten FW;Frantom PA
在Suf铁 - 硫簇组装途径中，半胱氨酸脱硫酶SufS的活性受与其辅助性硫转移酶蛋白SufE相互作用的调节。已表明SufE可刺激SufS活性，可能是通过诱导SufS活性位点的构象变化以促进脱硫步骤，以及在转硫醇化步骤中作为一种高效的过硫化物受体来实现的。先前的结果指向一种通过“半位点”机制进行的额外调节水平，当二聚体SufS在脱硫和转硫醇化活性之间转换时，该机制影响与SufE的化学计量和亲和力。通过主链酰胺氢/氘交换质谱法对SufS的共价过硫化物中间体进行研究，确定了两个活性位点肽段（残基225 - 236和356 - 366）以及SufS二聚体界面处的两个肽段（残基89 - 100和243 - 255），它们在中间体形成时氘摄取发生变化。这些肽段中的残基在过硫化物形成时被组织起来在两个活性位点之间形成一个通道，并且包括关键的跨单体相互作用，表明它们可能在半位点调节中起作用。通过丙氨酸扫描诱变对二聚体界面上的三个进化保守残基（R92、E96和E250）进行了研究。其中两种被替换的酶（E96A和E250A SufS）导致KSufE值增加了6倍，证实了其功能作用。对已报道的SufS和SufS同源物CsdA的晶体结构中的二聚体界面重新进行检查，发现了二聚体界面处先前未被注意到的残基移动性。通过氢/氘交换确定的二聚体界面构象变化，经诱变和结构报告证实，为SufS活性的半位点调节中的活性位点通讯提供了一种物理机制。鉴于界面相互作用的保守性，这种机制可能广泛适用于II型半胱氨酸脱硫酶系统。
In the Suf Fe-S cluster assembly pathway, the activity of the cysteine desulfurase, SufS, is regulated by interactions with the accessory sulfotransferase protein, SufE. SufE has been shown to stimulate SufS activity, likely through inducing conformational changes in the SufS active site that promote the desulfurase step and by acting as an efficient persulfide acceptor in the transpersulfuration step. Previous results point toward an additional level of regulation through a ‘half-sites’ mechanism that affects the stoichiometry and affinity for SufE as the dimeric SufS shifts between desulfurase and transpersulfuration activities. Investigation of the covalent persulfide intermediate of SufS by backbone amide hydrogen/deuterium exchange mass spectrometry identified two active site peptides (residues 225–236 and 356–366) and two peptides at the dimer interface of SufS (residues 89–100 and 243–255) that exhibit changes in deuterium uptake upon formation of the intermediate. Residues in these peptides are organized to form a conduit between the two active sites upon persulfide formation and include key cross-monomer interactions, suggesting they may play a role in the half-sites regulation. Three evolutionarily conserved residues at the dimer interface (R92, E96, and E250) were investigated by alanine scanning mutagenesis. Two of the substituted enzymes (E96A and E250A SufS) resulted in 6-fold increases in the value of KSufE, confirming a functional role. Re-examination of the dimer interface in reported crystal structures of SufS and the SufS-homolog CsdA identified previously unnoticed residue mobility at the dimer interface. The identification of conformational changes at the dimer interface by hydrogen/deuterium exchange confirmed by mutagenesis and structural reports provides a physical mechanism for active site communication in the half-sites regulation of SufS activity. Given the conservation of the interface interactions, this mechanism may be broadly applicable to type II cysteine desulfurase systems.
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影响因子：
4.8
作者：
Kim, Sunmin;Park, SangYoun
通讯作者：
Park, SangYoun
DOI：
10.1073/pnas.1702849114
发表时间：
2017-07-03
影响因子：
11.1
作者：
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影响因子：
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作者：
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影响因子：
2.9
作者：
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影响因子：
2.9
作者：
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