L-Ergothioneine (ET), the 2-thioimidazole derivative of trimethylhistidine, is biosynthesized by select fungi and bacteria, notably Mycobacterium tuberculosis, and functions as a scavenger of ...reactive oxygen species. The extent to which ET broadly functions in bacterial cells unable to synthesize it is unknown. Here we show that spd_1642-1643 in Streptococcus pneumoniae, a Gram-positive respiratory pathogen, encodes an ET uptake ATP-binding cassette (ABC) transporter, designated EgtU. The solute binding domain (SBD) of EgtU, EgtUC, binds ET with high affinity and exquisite specificity in a cleft between the two subdomains, with cation-π interactions engaging the betaine moiety and a network of water molecules that surround the thioimidazole ring. EgtU is highly conserved among known quaternary amine compound-specific transporters and widely distributed in Firmicutes, including the human pathogens Listeria monocytogenes, as BilEB, Enterococcus faecalis and Staphylococcus aureus. ET increases the chemical diversity of the low molecular weight thiol pool in Gram-positive human pathogens and may contribute to antioxidant defenses in the infected host.
Bacterial transition metal homoeostasis or simply 'metallostasis' describes the process by which cells control the intracellular availability of functionally required metal cofactors, from manganese ...(Mn) to zinc (Zn), avoiding both metal deprivation and toxicity. Metallostasis is an emerging aspect of the vertebrate host-pathogen interface that is defined by a 'tug-of-war' for biologically essential metals and provides the motivation for much recent work in this area. The host employs a number of strategies to starve the microbial pathogen of essential metals, while for others attempts to limit bacterial infections by leveraging highly competitive metals. Bacteria must be capable of adapting to these efforts to remodel the transition metal landscape and employ highly specialized metal sensing transcriptional regulators, termed metalloregulatory proteins,and metallochaperones, that allocate metals to specific destinations, to mediate this adaptive response. In this essay, we discuss recent progress in our understanding of the structural mechanisms and metal specificity of this adaptive response, focusing on energy-requiring metallochaperones that play roles in the metallocofactor active site assembly in metalloenzymes and metallosensors, which govern the systems-level response to metal limitation and intoxication.
Allosteric communication between two ligand-binding sites in a protein is a central aspect of biological regulation that remains mechanistically unclear. Here we show that perturbations in ...equilibrium picosecond–nanosecond motions impact zinc (Zn)-induced allosteric inhibition of DNA binding by the Zn efflux repressor CzrA (chromosomal zinc-regulated repressor). DNA binding leads to an unanticipated increase in methyl side-chain flexibility and thus stabilizes the complex entropically; Zn binding redistributes these motions, inhibiting formation of the DNA complex by restricting coupled fast motions and concerted slower motions. Allosterically impaired CzrA mutants are characterized by distinct nonnative fast internal dynamics “fingerprints” upon Zn binding, and DNA binding is weakly regulated. We demonstrate the predictive power of the wild-type dynamics fingerprint to identify key residues in dynamics-driven allostery. We propose that driving forces arising from dynamics can be harnessed by nature to evolve new allosteric ligand specificities in a compact molecular scaffold.
Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to ...specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.
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•The N terminus of ZNG1 interacts with the zinc finger of METAP1•ZNG1 promotes METAP1 activity in a GTP-hydrolysis- and Zn-dependent manner•Loss of ZNG1 impairs mitochondrial function and cellular proliferation•Zng1 mutant animals are sensitive to Zn starvation and inhibition of METAP activity
ZNG1 is a putative Zn metallochaperone that supports the activity of Zn-dependent proteins in vertebrates, thereby maintaining Zn homeostasis and cellular integrity.
The RNA helicase eIF4A plays a key role in unwinding of mRNA and scanning during translation initiation. Free eIF4A is a poor helicase and requires the accessory proteins eIF4G and eIF4H. However, ...the structure of the helicase complex and the mechanisms of stimulation of eIF4A activity have remained elusive. Here we report the topology of the eIF4A/4G/4H helicase complex, which is built from multiple experimentally observed domain-domain contacts. Remarkably, some of the interactions are continuously rearranged during the ATP binding/hydrolysis cycle of the helicase. We show that the accessory proteins modulate the affinity of eIF4A for ATP by interacting simultaneously with both helicase domains and promoting either the closed, ATP-bound conformation or the open, nucleotide-free conformation. The topology of the complex and the spatial arrangement of the RNA-binding surfaces offer insights into their roles in stimulation of helicase activity and the mechanisms of mRNA unwinding and scanning.
Abstract
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.
Streptococcus pneumoniae (pneumococcus) is a Gram-positive commensal and human respiratory pathogen. How this bacterium satisfies its nutritional iron (Fe) requirement in the context of endogenously ...produced hydrogen peroxide is not well understood. Here, we characterize a novel virulence-associated Rrf2-family transcriptional repressor that we term SifR (streptococcal IscR-like family transcriptional repressor) encoded by spd_1448 and conserved in Streptococci. Global transcriptomic analysis of a ΔsifR strain defines the SifR regulon as genes encoding a candidate catechol dioxygenase CatE, an uncharacterized oxidoreductase YwnB, a candidate flavin-dependent ferric reductase YhdA, a candidate heme-based ferric reductase domain–containing protein and the Piu (pneumococcus iron uptake) Fe transporter (piuBCDA). Previous work established that membrane-anchored PiuA binds FeIII–bis-catechol or monocatechol complexes with high affinity, including the human catecholamine stress hormone, norepinephrine. We demonstrate that SifR senses quinone via a single conserved cysteine that represses its regulon when in the reduced form. Upon reaction with catechol-derived quinones, we show that SifR dissociates from the DNA leading to regulon derepression, allowing the pneumococcus to access a catechol-derived source of Fe while minimizing reactive electrophile stress induced by quinones. Consistent with this model, we show that CatE is an FeII-dependent 2,3-catechol dioxygenase with broad substrate specificity, YwnB is an NAD(P)H-dependent quinone reductase capable of reducing the oxidized and cyclized norepinephrine, adrenochrome, and YhdA is capable of reducing a number of FeIII complexes, including PiuA-binding transport substrates. These findings are consistent with a model where FeIII–catechol complexes serve as significant nutritional Fe sources in the host.
Hydrogen sulfide (H2S) is both a lethal gas and an emerging gasotransmitter in humans, suggesting that the cellular H2S level must be tightly regulated. CstB is encoded by the cst operon of the major ...human pathogen Staphylococcus aureus and is under the transcriptional control of the persulfide sensor CstR and H2S. Here, we show that CstB is a multifunctional Fe(II)-containing persulfide dioxygenase (PDO), analogous to the vertebrate protein ETHE1 (ethylmalonic encephalopathy protein 1). Chromosomal deletion of ethe1 is fatal in vertebrates. In the presence of molecular oxygen (O2), hETHE1 oxidizes glutathione persulfide (GSSH) to generate sulfite and reduced glutathione. In contrast, CstB oxidizes major cellular low molecular weight (LMW) persulfide substrates from S. aureus, coenzyme A persulfide (CoASSH) and bacillithiol persulfide (BSSH), directly to generate thiosulfate (TS) and reduced thiols, thereby avoiding the cellular toxicity of sulfite. Both Cys201 in the N-terminal PDO domain (CstBPDO) and Cys408 in the C-terminal rhodanese domain (CstBRhod) strongly enhance the TS generating activity of CstB. CstB also possesses persulfide transferase (PT; reverse rhodanese) activity, which generates TS when provided with LMW persulfides and sulfite, as well as conventional thiosulfate transferase (TST; rhodanese) activity; both of these activities require Cys408. CstB protects S. aureus against H2S toxicity, with the C201S and C408S cstB genes being unable to rescue a NaHS-induced ΔcstB growth phenotype. Induction of the cst operon by NaHS reveals that functional CstB impacts cellular TS concentrations. These data collectively suggest that CstB may have evolved to facilitate the clearance of LMW persulfides that occur upon elevation of the level of cellular H2S and hence may have an impact on bacterial viability under H2S misregulation, in concert with the other enzymes encoded by the cst operon.
Transition metal homeostasis ensures that cells and organisms obtain sufficient metal to meet cellular demand while dispensing with any excess so as to avoid toxicity. In bacteria, zinc restriction ...induces the expression of one or more Zur (zinc-uptake repressor)-regulated Cluster of Orthologous Groups (COG) COG0523 proteins. COG0523 proteins encompass a poorly understood sub-family of G3E P-loop small GTPases, others of which are known to function as metallochaperones in the maturation of cobalamin (CoII) and NiII cofactor-containing metalloenzymes. Here, we use genomic enzymology tools to functionally analyse over 80 000 sequences that are evolutionarily related to Acinetobacter baumannii ZigA (Zur-inducible GTPase), a COG0523 protein and candidate zinc metallochaperone. These sequences segregate into distinct sequence similarity network (SSN) clusters, exemplified by the ZnII-Zur-regulated and FeIII-nitrile hydratase activator CxCC (C, Cys; X, any amino acid)-containing COG0523 proteins (SSN cluster 1), NiII-UreG (clusters 2, 8), CoII-CobW (cluster 4), and NiII-HypB (cluster 5). A total of five large clusters that comprise ≈ 25% of all sequences, including cluster 3 which harbors the only structurally characterized COG0523 protein, Escherichia coli YjiA, and many uncharacterized eukaryotic COG0523 proteins. We also establish that mycobacterial-specific protein Y (Mpy) recruitment factor (Mrf), which promotes ribosome hibernation in actinomycetes under conditions of ZnII starvation, segregates into a fifth SSN cluster (cluster 17). Mrf is a COG0523 paralog that lacks all GTP-binding determinants as well as the ZnII-coordinating Cys found in CxCC-containing COG0523 proteins. On the basis of this analysis, we discuss new perspectives on the COG0523 proteins as cellular reporters of widespread nutrient stress induced by ZnII limitation.
Staphylococcus aureus is a commensal pathogen that has evolved to protect itself from unfavorable conditions by forming complex community structures termed biofilms. The regulation of the formation ...of these structures is multifactorial and in S. aureus involves a number of transcriptional regulators. GbaA (glucose-induced biofilm accessory protein A) is a tetracycline repressor (TetR) family regulator that harbors two conserved Cys residues (C55 and C104) and impacts the regulation of formation of poly-N-acetylglucosamine-based biofilms in many methicillin-resistant S. aureus (MRSA) strains. Here, we show that GbaA-regulated transcription of a divergently transcribed operon in a MRSA strain can be induced by potent electrophiles, N-ethylmaleimide and methylglyoxal. Strikingly, induction of transcription in cells requires C55 or C104, but not both. These findings are consistent with in vitro small-angle X-ray scattering, chemical modification, and DNA operator binding experiments, which reveal that both reduced and intraprotomer (C55–C104) disulfide forms of GbaA have very similar overall structures and each exhibits a high affinity for the DNA operator, while DNA binding is strongly inhibited by derivatization of one or the other Cys residues via formation of a mixed disulfide with bacillithiol disulfide or a monothiol derivatization adduct with NEM. While both Cys residues are reactive toward electrophiles, C104 in the regulatory domain is the more reactive thiolate. These characteristics enhance the inducer specificity of GbaA and would preclude sensing of generalized cellular oxidative stress via disulfide bond formation. The implications of the findings for GbaA function in MRSA strains are discussed.
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