Protocol for using organic persulfides to measure the chemical reactivity of persulfide sensors

Hydrogen sulfide (H2S) and downstream reactive sulfur species (RSS), including organic persulfides, protect bacterial cells against diverse oxidative stressors. Specialized dithiol-based transcriptional repressors sense persulfides directly to control cellular H2S/RSS and avoid toxicity. Here, we present a protocol to quantify the kinetics of chemical reactivity of cysteines in two bacterial persulfide sensors toward cysteine persulfide and glutathione persulfide, with a LC-ESI-MS analysis that results in a kinetic model. This protocol has potential applications to other cysteine-containing proteins and oxidants. For complete details on the use and execution of this protocol, please refer to Fakhoury et al. (2021) and Capdevila et al. (2021).Fil: Fakhoury, Joseph N.. Indiana University; Estados UnidosFil: Capdevila, Daiana Andrea. Indiana University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Giedroc, David Peter. Indiana University; Estados Unido

Functional asymmetry and chemical reactivity of CsoR family persulfide sensors

CstR is a persulfide-sensing member of the functionally diverse copper-sensitive operon repressor (CsoR) superfamily. While CstR regulates the bacterial response to hydrogen sulfide (H2S) and more oxidized reactive sulfur species (RSS) in Gram-positive pathogens, other dithiol-containing CsoR proteins respond to host derived Cu(I) toxicity, sometimes in the same bacterial cytoplasm, but without regulatory crosstalk in cells. It is not clear what prevents this crosstalk, nor the extent to which RSS sensors exhibit specificity over other oxidants. Here, we report a sequence similarity network (SSN) analysis of the entire CsoR superfamily, which together with the first crystallographic structure of a CstR and comprehensive mass spectrometry-based kinetic profiling experiments, reveal new insights into the molecular basis of RSS specificity in CstRs. We find that the more N-terminal cysteine is the attacking Cys in CstR and is far more nucleophilic than in a CsoR. Moreover, our CstR crystal structure is markedly asymmetric and chemical reactivity experiments reveal the functional impact of this asymmetry. Substitution of the Asn wedge between the resolving and the attacking thiol with Ala significantly decreases asymmetry in the crystal structure and markedly impacts the distribution of species, despite adopting the same global structure as the parent repressor. Companion NMR, SAXS and molecular dynamics simulations reveal that the structural and functional asymmetry can be traced to fast internal dynamics of the tetramer. Furthermore, this asymmetry is preserved in all CstRs and with all oxidants tested, giving rise to markedly distinct distributions of crosslinked products. Our exploration of the sequence, structural, and kinetic features that determine oxidant-specificity suggest that the product distribution upon RSS exposure is determined by internal flexibility.Fil: Fakhoury, Joseph N. Indiana University; Estados UnidosFil: Zhang, Yifan. Indiana University; Estados UnidosFil: Edmonds, Katherine A. Indiana University; Estados UnidosFil: Bringas, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Luebke, Justin L. Indiana University; Estados UnidosFil: Gonzalez Gutierrez, Giovanni. Indiana University; Estados UnidosFil: Capdevila, Daiana Andrea. Indiana University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Giedroc, David Peter. Indiana University; Estados Unido

The global structures of a wild-type and poorly functional plant luteoviral mRNA pseudoknot are essentially identical

The helical junction region of a −1 frameshift stimulating hairpin-type mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) is characterized by a novel C27·(G7–C14) loop 2–stem 1 minor groove base triple, which is stacked on a C8(+)·(G12–C28) loop 1–stem 2 major groove base triple. Substitution of C27 with adenosine reduces frameshifting efficiency to a level just twofold above the slip-site alone. Here, we show that the global structure of the C27A ScYLV RNA is nearly indistinguishable from the wild-type counterpart, despite the fact that the helical junction region is altered and incorporates the anticipated isostructural A27·(G7–C14) minor groove base triple. This interaction mediates a 2.3-Å displacement of C8(+) driven by an A27 N6–C8(+) O2 hydrogen bond as part of an A((n−1))·C(+)·G-C(n) base quadruple. The helical junction regions of the C27A ScYLV and the beet western yellows virus (BWYV) pseudoknots are essentially superimposable, the latter of which contains an analogous A25·(G7–C14) minor groove base triple. These results reveal that the global ground-state structure is not strongly correlated with frameshift stimulation and point to a reduced thermodynamic stability and/or enhanced kinetic lability that derives from an altered helical junction architecture in the C27A ScYLV RNA as a significant determinant for setting frameshifting efficiencies in plant luteoviral mRNA pseudoknots

Multi-metal nutrient restriction and crosstalk in metallostasis systems in microbial pathogens

Transition metals from manganese to zinc function as catalytic and structural cofactors for an amazing diversity of proteins and enzymes, and thus are essential for all forms of life. During infection, inflammatory host proteins limit the accessibility of multiple transition metals to invading pathogens in a process termed nutritional immunity. In order to respond to host-mediated metal starvation, bacteria employ both protein and RNA-based mechanisms to sense prevailing transition metal concentrations that collectively regulate systems-level strategies to maintain cellular metallostasis. In this review, we discuss a number of recent advances in our understanding of how bacteria orchestrate the adaptive response to host-mediated multi-metal restriction, highlighting crosstalk among these regulatory systems.Fil: Jordan, Matthew R.. Indiana University; Estados UnidosFil: Wang, Jiefei. Indiana University; Estados UnidosFil: Capdevila, Daiana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Giedroc, David Peter. Indiana University; Estados Unido

Molecular Evolution of Transition Metal Bioavailability at the Host–Pathogen Interface

The molecular evolution of the adaptive response at the host–pathogen interface has been frequently referred to as an 'arms race' between the host and bacterial pathogens. The innate immune system employs multiple strategies to starve microbes of metals. Pathogens, in turn, develop successful strategies to maintain access to bioavailable metal ions under conditions of extreme restriction of transition metals, or nutritional immunity. However, the processes by which evolution repurposes or re-engineers host and pathogen proteins to perform or refine new functions have been explored only recently. Here we review the molecular evolution of several human metalloproteins charged with restricting bacterial access to transition metals. These include the transition metal-chelating S100 proteins, natural resistance-associated macrophage protein-1 (NRAMP-1), transferrin, lactoferrin, and heme-binding proteins. We examine their coevolution with bacterial transition metal acquisition systems, involving siderophores and membrane-spanning metal importers, and the biological specificity of allosteric transcriptional regulatory proteins tasked with maintaining bacterial metallostasis. We also discuss the evolution of metallo-β-lactamases; this illustrates how rapid antibiotic-mediated evolution of a zinc metalloenzyme obligatorily occurs in the context of host-imposed nutritional immunity.Fil: Antelo, Giuliano Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Giedroc, David Peter. Indiana University; Estados UnidosFil: Capdevila, Daiana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Sensing and regulation of reactive sulfur species (RSS) in bacteria

The infected host deploys generalized oxidative stress caused by small inorganic reactive molecules as antibacterial weapons. An emerging consensus is that hydrogen sulfide (H2S) and forms of sulfur with sulfur–sulfur bonds termed reactive sulfur species (RSS) provide protection against oxidative stressors and antibiotics, as antioxidants. Here, we review our current understanding of RSS chemistry and its impact on bacterial physiology. We start by describing the basic chemistry of these reactive species and the experimental approaches developed to detect them in cells. We highlight the role of thiol persulfides in H2S-signaling and discuss three structural classes of ubiquitous RSS sensors that tightly regulate cellular H2S/RSS levels in bacteria, with a specific focus on the chemical specificity of these sensors.Fil: Giedroc, David Peter. Indiana University; Estados UnidosFil: Antelo, Giuliano Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Fakhoury, Joseph N.. Indiana University; Estados UnidosFil: Capdevila, Daiana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Mechanistic Insights into the Metal-Dependent Activation of ZnII^{\rm{II}}-Dependent Metallochaperones

Members of the COG0523 subfamily of candidate GTPase metallochaperones function in bacterial transition-metal homeostasis, but the nature of the cognate metal, mechanism of metal transfer, and identification of target protein(s) for metal delivery remain open questions. Here, we explore the multifunctionality of members of the subfamily linked to delivering Zn$^{\rm{II}}$ to apoprotein targets under conditions of host-imposed transition-metal depletion. We examine two zinc-uptake repressor (Zur)-regulated COG0523 family members, each from a major human pathogen, Acinetobacter baumannii (AbZigA) and Staphylococcus aureus (SaZigA), in an effort to develop a model for Zn$^{\rm{II}}$ metallochaperone activity. Zn$^{\rm{II}}$ chelator competition experiments reveal one high-affinity (K$_{\rm{Zn1}}$ ≈ 10$^{10}$–10$^{11}$ M$^{–1}$) metal-binding site in each GTPase, while AbZigA and SaZigA are characterized by an additional one and two (lower-affinity) metal-binding sites, respectively. CoII titrations reveal that both metallochaperones have similar electronic absorption characteristics that indicate the presence of two tetrahedral metal coordination sites. High-affinity metal binding at the CXCC motif activates the GTPase activity of both enzymes, with Zn$^{\rm{II}}$ more effective than Co$^{\rm{II}}$. Both GTPases bind the product, GDP, more tightly in the apoprotein than the Zn$^{\rm{II}}$-bound state and exhibit what is best described as a “locked” conformation around the GTP substrate. Negative thermodynamic linkage is observed between nucleotide binding and metal binding, leading to a new mechanistic model for COG0523-catalyzed metal delivery

A Mn-sensing riboswitch activates expression of a Mn2+/Ca2+ ATPase transporter in Streptococcus

Maintaining manganese (Mn) homeostasis is important for the virulence of numerous bacteria. In the human respiratory pathogen $Streptococcus pneumoniae$, the Mn-specific importer PsaBCA, exporter MntE, and transcriptional regulator PsaR establish Mn homeostasis. In other bacteria, Mn homeostasis is controlled by $yybP-ykoY$ family riboswitches. Here, we characterize a $yybP-ykoY$ family riboswitch upstream of the $mgtA$ gene encoding a P$_{\rm{II}}$-type ATPase in $S. pneumoniae$, suggested previously to function in Ca$^{2+}$ efflux. We show that the $mgtA$ riboswitch aptamer domain adopts a canonical $yybP-ykoY$ structure containing a three-way junction that is compacted in the presence of Ca$^{2+}$ or Mn$^{2+}$ at a physiological Mg$^{2+}$ concentration. Although Ca$^{2+}$ binds to the RNA aptamer with higher affinity than Mn$^{2+}$, in vitro activation of transcription read-through of $mgtA$ by Mn$^{2+}$ is much greater than by Ca$^{2+}$. Consistent with this result, $mgtA$ mRNA and protein levels increase ≈5-fold during cellular Mn stress, but only in genetic backgrounds of $S. pneumoniae$ and $Bacillus subtilis$ that exhibit Mn$^{2+}$ sensitivity, revealing that this riboswitch functions as a failsafe ‘on’ signal to prevent Mn$^{2+}$ toxicity in the presence of high cellular Mn$^{2+}$. In addition, our results suggest that the $S. pneumoniae$ $yybP-ykoY$ riboswitch functions to regulate Ca$^{2+}$ efflux under these conditions