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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 thiosulfate- treated 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.
Hydrogen sulfide (H2S), along with nitric oxide (NO) and carbon monoxide (CO), is an important gasotransmitter and plays an essential role in cell physiology by signaling through sulfhydration of cysteine residues in proteins. 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 (S2O32-) to cyanide (CN-) that is important for detoxification of cells. 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-). The most well-studied rhodanese domain is that of bovine liver rhodanese. 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. 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. 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. These results have been independently confirmed in another study of YgaP. 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. It has been known that S-nitrosylation and S-sulfhydration are mutually inhibitory processes, which may play important roles in H2S/NO signaling. 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. Therefore, it is important to understand the effects of these posttranslational modifications at atomic resolution. In this paper, we further investigated 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.
The main objective of this study is to characterize the structural changes, if any, that are induced in the YgaP rhodanese domain upon S-sulfhydration.
The fold of the rhodanese domain of YgaP treated with thiosulfate is essentially the same as that of unmodified protein (Fig. 1A). Our previous structural studies of this domain showed that the catalytic cysteine C63 of the protein prepared with 1,4-dithiothreitol (DTT) is partially S-nitrosylated, while the same cysteine in the structure of the protein prepared without DTT is both S-nitrosylated and S-sulfhydrated. Furthermore, the S-sulfydrated cysteine was found to adopt two conformations: one perpendicular to the plane of the catalytic loop and the second pointing toward T69 of the α4 helix (Fig. 1B). Here, we show that when the crystals of the protein prepared without DTT are treated with sodium thiosulfate, the catalytic cysteine is primarily S-sulfhydrated and the additional sulfur is pointing towards T69 of the α4 helix (Fig. 1C). Even though the overall protein fold remains the same, the S-sulfhydration of C63 with SH pointing towards T69 appears to cause the destabilization of the N-terminal part of the α4 helix in a progressive manner. This conclusion is based on the following observations: In the structure of the protein prepared with DDT, there is no S-sulfhydration and the α4 helix is well-defined, as illustrated by an electron density that is similar throughout the structure (Fig. 1A). However, when the protein was prepared without DTT, the S-sulfhydration with SH pointing towards T69 is present and the electron density is weaker for the first two turns of the α4 helix (Fig. 1B). Finally, the thiosulfate treatment of a protein crystal prepared without DTT leads to a higher occupancy of the SH pointing towards T69 and the electron density of the first two turns of the α4 helix are even weaker (Fig. 1C). The progressive destabilization of this part of the helix can also be illustrated quantitatively with B-factors. The average B-factors of the backbone atoms of the first two turns of helix α4 (i.e., residues 67–72) in the structure of the protein prepared with DTT, the structure of the protein prepared without DTT, and the structure from the crystal treated with thiosulfate are progressively increasing with values of 22.69, 25.11, and 32.80, respectively, whereas the average B-factor of the last two turns of this helix (i.e., residues 74–79) remain similar with values of 13.93, 16.00, and 14.77, respectively. The induction of an S-sulfhydration-dependent dynamical destabilization of the α4 helix is also supported by previous NMR titration studies. The T69 cross-peak in [15N, 1H]-TROSY spectra undergoes a significant broadening during the titration of the rhodanese domain with 1-4 mM sodium thiosulfate. S-sulfhydration-induced line broadening indicates conformational exchange dynamics of the N-terminal part of the α4 helix (Fig. 1G). In line with these interpretations and findings, the further treatment of the sample with 1–4 mM potassium cyanide, which causes the removal of the persulfide, shows a line narrowing of the T69 cross-peak in [15N, 1H]-TROSY spectra (Fig. 1G).
The catalytic loop of the YgaP rhodanese domain (residues 63–68) is characteristic of rhodaneses and overlaps very well with those from other rhodaneses. The N-H moieties of the loop are pointing to the center of the loop, where the SH sulfur atom of the S-sulfhydrated cysteine is expected to be based on the x-ray structures of bovine liver rhodanese and GlpE. The N-H dipoles create thereby a positively charged pocket in which the reactive negatively charged sulfur can be stabilized. In line with this interpretation, the crystal structure of the YgaP rhodanese domain prepared without DTT showed an electron density in this positively charged pocket at C63, which could be modeled as SH. Moreover, there is an additional electron density, which we attributed to NO from S-nitrosylation, since this electron density is strengthened when the crystals are soaked with the NO donor, S-nitrosocysteine (SNOC). Furthermore, there is yet an additional electron density pointing towards T69 of the α4 helix, suggesting yet another modification of C63 (Fig. 1B). Initially we thought that this is an alternative configuration of NO from S-nitrosylation. However, the current study of the structure of the crystal treated with thiosulfate shows that as the result of this treatment the electron density at C63 pointing towards T69 is indeed significantly more pronounced and accompanied with a corresponding loss of the electron density of helix α4 (Fig. 1F). This result, on the other hand, is against the expectation that the sulfur introduced by S-sulfhydration should be perpendicular to the plane of the catalytic loop as observed in other rhodaneses. Also peculiar is the transient nature of S-sulfhydration of YgaP. In an attempt to correlate the two unusual properties of YgaP, it can be speculated that this atypical configuration of S-sulfhydration pointing to the N-terminus of the helix α4 destabilizes the helix by weakening its dipole, inducing the dynamical interplay between the helix α4 and S-sulfhydration that makes the CYS-SH bond transient. Unfortunately, without knowing the physiological role of YgaP, it is not possible to annotate the biochemical processes that accompany the transient nature of S-sulfhydration and the interplay between S-sulfhydration and the helical dynamics.
Preparation of the rhodanese domain of YgaP (residues 1-109) were conducted as described in. Crystallization trials were conducted with several commercial screening kits, Crystal Screen, Crystal Screen 2 (Hampton Research, CA), Wizard Screens I and II (Emerald BioStructures, WA), PEG/ION and Nextal Classics suite (Qiagen, Inc., CA) using the Mosquito crystallization robot (TTP Labtech, MA). The trials yielded several crystallization hits, which were subsequently optimized. The final crystal was obtained using the hanging-drop vapor diffusion method, in which 1 µl of 10 mg/ml protein was mixed with 1 µl of a reservoir solution of 0.2 M sodium acetate in 0.1 M Tris-HCl pH 8.5 and 30% PEG 4k. Crystals appeared after a few days at 15°C and were frozen in liquid N2. To induce S-sulfhydration, crystals were soaked in 1 mM sodium thiosulfate for 5 min before freezing. The x-ray data of the crystals were collected at SSRL on the beamline 12–2 to 1.66Å resolution and processed by HKL2000. The structure was solved by molecular replacement (MR) using the structure from the untreated protein as a model. Model building was done in COOT version 0.8.1. The refinement of the structures was completed using REFMAC, version 5.8.0124. The structure figures were generated using Molscript version 2.1.2. The data processing and refinement statistics are:
Space group P3 (1)
Unit-cell parameters a (Å) 43.70 c (Å) 52.28
Resolution range (Å) 37.84-1.66 (1.75-1.66)
No. of observed reflections 70140
No. of unique reflections 13126
I/σ (I) 34.4 (16.2)
Redundancy 5.3 (4.2)
Completeness (%) 98.4 (89.3)
Rmerge 0.037 (0.057)
No. of atoms 995
No. of solvent atoms 147
No. of residues 103
r.m.s. bonds 0.009
Average B-factor (Å2) 16.96
main chain 14.10
side chain 19.07
Ramachandran favored (%) 93.3
allowed (%) 6.7
outliers (%) 0