Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator

Abstract

1Cysteine thiol-based transcriptional regulators orchestrate coordinated regulation of redox homeostasis 2 and other cellular processes by "sensing" or detecting a specific redox-active molecule, which in turn 3 activates the transcription of a specific detoxification pathway. The extent to which these sensors are 4 truly specific in cells for a singular class of reactive small molecule stressors, e.g., reactive oxygen or 5 sulfur species, is largely unknown. Here we report novel structural and mechanistic insights into a thiol-6 based transcriptional repressor SqrR, that reacts exclusively with organic and inorganic oxidized sulfur 7 species, e.g., persulfides, to yield a unique tetrasulfide bridge that allosterically inhibits DNA operator-8 promoter binding. Evaluation of five crystallographic structures of SqrR in various derivatized states, 9 coupled with the results of a mass spectrometry-based kinetic profiling strategy, suggest that persulfide 1 0 selectivity is determined by structural frustration of the disulfide form. This energetic roadblock 1 1 effectively decreases the reactivity toward major oxidants to kinetically favor formation of the 1 2 tetrasulfide product. These findings lead to the identification of an uncharacterized repressor from the 1 3 increasingly antibiotic-resistant bacterial pathogen, Acinetobacter baumannii, as a persulfide sensor, 1 4illustrating the predictive power of this work and potential applications to bacterial infectious disease. 1 5 1 6Regulatory posttranslational modifications on thiol groups of redox-active and allosterically important 1 cysteines are a critical component of signaling and redox regulation in cells 1-3 . The successful 2 adaptation of microorganisms to an ever-changing microenvironment depends, to a considerable degree, 3 on the specificity of the regulation of the expression of needed repair enzymes 4-6 . Understanding the 4 extent to which transcriptional regulation is specific to a particular redox-active small molecule(s) is 5 complicated by cross-talk or interconversion among these species in cells 7,8 and the difficulties 6 associated with identifying a posttranslational modification that is biologically meaningful at cell-7 appropriate concentrations that induce a cellular response 9 . Prominent exceptions to this are a handful 8 of transcriptional regulators that employ a metallated cofactor, such as a mononuclear iron 10,11 , an iron-9 sulfur cluster 12,13 or a heme-based sensing moeity 14 ; here, the local inorganic chemistry alone is 1 0 typically sufficient to explain the specificity of these switches toward their inducer 15 . In fact, it remains 1 1 unclear if and how a non-metallated, dithiol-based transcriptional regulator achieves a similar level of 1 2 specificity toward different cellular oxidants.

H2S and reactive sulfur signaling at the host-bacterial pathogen interface

“…Although the connection between biofilm regulation and H 2 S/RSS homeostasis is largely speculation at this point, H 2 S has been detected in cystic fibrosis sputum, a complex biofilm (111), and H 2 S has been found to promote formation of biofilms by intestinal microbiota while reducing the proliferation of planktonic bacterial cells (112). We recently characterized the biofilm growth-associated repressor, BigR, in A. baumannii as an RSS sensor (20,113), as previously characterized in plant pathogens (114)(115)(116). That work also identified two transcriptional regulators in A. baumannii known or projected to be involved in biofilm regulation that were characterized by significantly increased protein persulfidation mediated by exogenous sulfide (20).…”

“…These RSS sensors are widespread and have been identified in both Gram-positive and Gram-negative organisms. They include CstR from S. aureus (15,16) and E. faecalis (17), SqrR from Rhodobacter capsulatus (18,113), the SqrR homolog BigR from Xylella fastidiosa (114)(115)(116) and A. baumannii (20), and FisR from Cupriavidus pinatubonensis (19) and A. baumannii (20).…”

“…SqrR adopts the ArsR family a1-a2-a3-a4-b1-b2-a5 "winged-helical" dimeric fold, where one Cys in the a2 helix and one Cys in the a5 helix from the same subunit create a pair of dithiol RSS-sensing sites on the dimer (Fig. 5B, middle) (113). Other RSS-responsive ArsR family repressors include the biofilm growth-associated repressor BigR, characterized in X. fastidiosa (114)(115)(116) and A. baumannii (20), and PigS characterized in Serratia spp.…”

“…The ArsR-family RSS sensors that have been functionally characterized behave analogously to CstR, functioning as repressors in the reduced state and dissociating from the DNA upon reaction with sulfane sulfur-containing RSS, to readily form nearly exclusively tetrasulfide (SqrR) and pentasulfide (BigR) bridges, respectively (Fig. 5B, right) (113). In contrast to CstR, these (poly)sulfur bridges are formed within a subunit, and although other linkages are made, they are far less abundant compared with the mixture of products found in CstR (16).…”

“…Recent work from our laboratory utilized SqrR as a model dithiol transcriptional regulator to investigate the structural and reactivity features that govern its oxidant selectivity and specificity (113). Indeed, SqrR is specific for sulfane sulfur and only forms a disulfide when treated with potent diazirene electrophile TMAD (diamide), but not with more common cellular oxidants including GSH disulfide or H 2 O 2 .…”