Varatnitskaya Marharyta, Fasel Julia, Müller Alexandra, Lupilov Natalie, Shi Yunlong, Fuchs Kristin, Krewing Marco, Jung Christoph, Jacob Timo, Sitek Barbara, Bandow Julia E, Carroll Kate S, Hofmann Eckhard, Leichert Lars I
Ruhr University Bochum, Institute of Biochemistry and Pathobiochemistry, Microbial Biochemistry, Bochum, Germany.
UF Scripps Biomedical Research, Department of Chemistry, 130 Scripps Way, Jupiter, FL, 33458, USA.
Redox Biol. 2022 Jul;53:102332. doi: 10.1016/j.redox.2022.102332. Epub 2022 May 7.
Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli's proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidA, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSOH) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidA's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidA and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.
在生理条件下，大肠杆菌RidA是一种烯胺/亚胺脱氨酶，可促进活性烯胺/亚胺中间体释放氨。然而，当被次氯酸（HOCl）修饰时，它会变成一种强大的伴侣样保持酶，在氧化应激期间能有效保护大肠杆菌的蛋白质组。然而，尚不清楚哪些残基需要被氯化才能激活。在这里，我们结合液相色谱-串联质谱分析、化学蛋白质组学方法和诱变研究，以确定负责RidA伴侣样功能的残基。通过对消化后的RidA进行液相色谱-串联质谱分析，我们获得了一个精氨酸残基被氯化的直接证据。为了克服N-氯胺修饰的不稳定性，我们建立了一种化学蛋白质组学方法，使用5-（二甲基氨基）萘-1-亚磺酸（DANSOH）作为探针来标记N-氯化赖氨酸。使用该探针，我们能够检测到另外六个赖氨酸残基的N-氯化。此外，通过诱变研究对单个精氨酸和赖氨酸残基的作用进行基因探究，我们发现去除精氨酸R105和/或R128会导致RidA伴侣活性大幅降低。这些结果与结构分析一起证实，RidA的伴侣活性与蛋白质表面正电荷的丧失同时发生，导致蛋白质整体疏水性增加。RidA的分子建模以及即使在没有HOCl的情况下仍具有伴侣活性的RidA变体的合理设计进一步支持了我们的假设。我们的数据为RidA以及越来越多其他HOCl激活的伴侣中发现的HOCl介导的伴侣活性提供了分子机制。
