S-Sulfhydration of the Catalytic Cysteine in the Rhodanese Domain of YgaP is Complex Dynamic Process

The S-sulfhydration of cysteine residues in proteins has emerged as a common modification that can modulate the activity of a protein. The ubiquity of the rhodanese domain and its occurrence in a wide variety of protein families indicates that it has diverse roles in physiology. Contrary to common expectations, previous structural studies of several rhodanese domains concluded that S-sulfhydration does not induce a structural change in the protein. The presented x-ray structure of a thiosulfatetreated crystal of the rhodanese domain of the E. coli integral membrane protein YgaP reveals two important findings: (1) The S-sulfhydrated catalytic cysteine C63 adopts an atypical conformation. (2) S-sulfhydration leads to a destabilization of the N-terminal part of the helix adjacent to the catalytic loop. These findings assert that S-sulfhydration is accompanied by a specific and complex dynamic process. Introduction Hydrogen sulfide (H2S), along with nitric oxide (NO) and carbon monoxide (CO), is an important gasotransmitter [1] [2] and plays an essential role in cell physiology by signaling through sulfhydration of cysteine residues in proteins [3]. Rhodaneses/sulfurtransferases form a group of enzymes widely distributed in prokaryotic and eukaryotic cells that via sulfhydration are able to catalyze the transfer of sulfur from thiosulfate (S2O3) to cyanide (CN-) that is important for detoxification of cells [4]. The catalysis is a two-step reaction in which the thiol group of the cysteine first reacts with the thiosulfate anion to form an enzyme-persulfide intermediate (CYS-SH), which then reacts with the cyanide ion to produce the much less toxic thiocyanate (SCN-) [4]. The most well-studied rhodanese domain is that of bovine liver rhodanese [5] [6] [7] [8]. The active cysteine is located in the cradle-shaped catalytic loop formed by the backbone atoms of the loop residues in such a way that the reactive sulfur, most likely negatively charged, is pointing to the center of the cradle. It has been stipulated that the S-sulfhydrated enzyme undergoes a significant conformational change. However, the crystal structures of the sulfur-free as well as S-sulfhydrated enzyme are very similar [9]. Even more puzzling results came from the study that investigated in detail the impact of S-sulfhydration on the structure and dynamics of the rhodanese domain of the E. coli integral membrane protein YgaP using solution NMR as the main technique [10]. The study revealed that the S-sulfhydration of YgaP is a transient process: A titration with 1–4 mM sodium thiosulfate to the solution containing 13C, 15N–labeled YgaP rhodanese domain revealed that rather than two distinct sets of cross-peaks corresponding to the S-bound and -unbound states, a single set of cross-peaks that shift upon titration is observed in two-dimensional [15N, 1H]-TROSY experiments indicating a fast exchange (i.e., in the micro– to millisecond range) between the S-bound and -unbound states [10]. These results have been independently confirmed in another study of YgaP [11]. Despite this peculiar finding, the crystal structure of the rhodanease domain of YgaP revealed that the active loop superimposes very well with the analogous bovine rhodanese loop. Furthermore, the structure also revealed that the protein overexpressed in E. coli is partially both S-sulfhydrated and S-nitrosylated and that the S-sulfhydration is likely present in two conformations [12]. It has been known that S-nitrosylation and S-sulfhydration are mutually inhibitory processes, which may play important roles in H2S/NO signaling [13]. It has also been suspected that the ubiquity and frequent presence of the rhodanese domains in multidomain proteins implies physiological functions other than cell detoxification [14]. Therefore, it is important to understand the effects of these posttranslational modifications at atomic resolution. In this paper, we further inS-sulfhydration of the catalytic cysteine in the rhodanese domain of YgaP is complex dynamic process DOI: 10.19185/matters.2016024 Matters (ISSN: 2297-8240) | 2 vestigated the impact of S-sulfhydration on the rhodanese domain of the E. coli integral membrane protein YgaP and found that S-sulfhydration triggers a dynamical process by destabilizing the α4 helix in the domain. Objective Themain objective of this study is to characterize the structural changes, if any, that are induced in the YgaP rhodanese domain upon S-sulfhydration.