Kim Dokyong, Singh Harsimran, Dai Yuyuan, Dong Guangchao, Busenlehner Laura S, Outten F Wayne, Frantom Patrick A
Department of Chemistry and Biochemistry , The University of Alabama , Tuscaloosa , Alabama 35487 , United States.
Department of Chemistry and Biochemistry , The University of South Carolina , Columbia , South Carolina 29208 , United States.
Biochemistry. 2018 Sep 4;57(35):5210-5217. doi: 10.1021/acs.biochem.7b01275. Epub 2018 Apr 5.
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 by 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 88-100 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 K, confirming a functional role. Re-examination of the dimer interface in reported crystal structures of SufS and the SufS homologue 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.
在Suf铁硫簇组装途径中，半胱氨酸脱硫酶SufS的活性受其与辅助硫转移酶蛋白SufE相互作用的调控。研究表明，SufE可能通过诱导SufS活性位点的构象变化来促进脱硫酶步骤，并在转硫步骤中作为有效的过硫化物受体，从而刺激SufS的活性。先前的研究结果表明，通过一种“半位点”机制存在额外的调控水平，该机制会影响二聚体SufS在脱硫酶和转硫活性之间转换时对SufE的化学计量和亲和力。通过主链酰胺氢氘交换质谱对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（225 - 236位和356 - 366位残基）以及SufS二聚体界面处的两个肽段（88 - 100位和243 - 255位残基），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会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，这表明它们可能在半位点调控中发挥作用。通过丙氨酸扫描诱变研究了二聚体界面处三个进化保守残基（R92、E96和E250）。其中两个取代酶（E96A和E250A SufS）的K值增加了6倍，证实了其功能作用。重新检查已报道的SufS晶体结构以及SufS同源物CsdA的二聚体界面，发现了先前未注意到的二聚体界面处的残基流动性。通过诱变和结构报告证实的氢氘交换确定的二聚体界面处的构象变化，为SufS活性的半位点调控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。
