Department of Environmental Toxicology, University of California, Davis, California 95616, United States.
J Proteome Res. 2010 Dec 3;9(12):6740-51. doi: 10.1021/pr100920r. Epub 2010 Nov 2.
Sulfur trafficking systems are multiprotein systems that synthesize sulfur-containing cofactors such as iron-sulfur clusters. The sulfur is derived enzymatically from cysteine and transferred between nucleophilic cysteine residues within proteins until incorporation into the relevant cofactor. As these systems are poorly understood, we have developed an in vivo method for characterizing these interactions and have applied our method to the SUF system of Escherichia coli, which is responsible for iron-sulfur cluster biogenesis under oxidative stress and iron limitation. Proteins that interact covalently with SufE were trapped in vivo, purified, and identified by mass spectrometry. We identified SufE-SufS and SufE-SufB interactions, interactions previously demonstrated in vitro, indicating that our method has the ability to identify physiologically relevant interactions. The sulfur acceptor function of SufE is likely due to the low pK(a) of its active site C51, which we determined to be 6.3 ± 0.7. We found that SufE interacts with several Fe-S cluster proteins, further supporting the validity of the method, and with tryptophanase, glutaredoxin-3, and glutaredoxin-4, possibly suggesting a role for these enzymes in iron-sulfur biogenesis by the SUF system. Our results indicate that this method could serve as a general tool for the determination of sulfur trafficking mechanisms.
硫转运系统是多蛋白系统，可合成含硫辅因子，如铁硫簇。硫通过半胱氨酸酶促合成，并在蛋白质内部的亲核半胱氨酸残基之间转移，直到掺入相关的辅因子中。由于这些系统尚未得到充分理解，我们开发了一种用于表征这些相互作用的体内方法，并将我们的方法应用于大肠杆菌的 SUF 系统，该系统负责在氧化应激和铁限制下进行铁硫簇生物发生。与 SufE 共价相互作用的蛋白质在体内被捕获、纯化，并通过质谱鉴定。我们鉴定了 SufE-SufS 和 SufE-SufB 相互作用，这是先前在体外证明的相互作用，表明我们的方法有能力识别生理相关的相互作用。SufE 的硫受体功能可能归因于其活性位点 C51 的低 pK(a)，我们确定其值为 6.3±0.7。我们发现 SufE 与几种 Fe-S 簇蛋白相互作用，这进一步支持了该方法的有效性，并且与色氨酸酶、谷氧还蛋白-3 和谷氧还蛋白-4 相互作用，可能表明这些酶在 SUF 系统的铁硫生物发生中发挥作用。我们的结果表明，该方法可以作为确定硫转运机制的通用工具。
