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.