Nitrate- and Nitrite-Sensing Histidine Kinases: Function, Structure, and Natural Diversity.
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ABSTRACT:
SUBMITTER: Gushchin I
PROVIDER: S-EPMC8199190 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Integral to the protein structure/function paradigm, oligomeric state is typically conserved along with function across evolution. However, notable exceptions such as the hemoglobins show how evolution can alter oligomerization to enable new regulatory mechanisms. Here, we examine this linkage in histidine kinases (HKs), a large class of widely distributed prokaryotic environmental sensors. While the majority of HKs are transmembrane homodimers, members of the HWE/HisKA2 family can deviate from this architecture as exemplified by our finding of a monomeric soluble HWE/HisKA2 HK (EL346, a photosensing light-oxygen-voltage [LOV]-HK). To further explore the diversity of oligomerization states and regulation within this family, we biophysically and biochemically characterized multiple EL346 homologs and found a range of HK oligomeric states and functions. Three LOV-HK homologs are primarily dimeric with differing structural and functional responses to light, while two Per-ARNT-Sim-HKs interconvert between differentially active monomers and dimers, suggesting dimerization might control enzymatic activity for these proteins. Finally, we examined putative interfaces in a dimeric LOV-HK, finding that multiple regions contribute to dimerization. Our findings suggest the potential for novel regulatory modes and oligomeric states beyond those traditionally defined for this important family of environmental sensors.
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Project description:Two-component signal transduction systems (TCSs) are widely conserved in bacteria to respond to and adapt to the changing environment. Since TCSs are also involved in controlling the expression of virulence, biofilm formation, quorum sensing, and antimicrobial resistance in pathogens, they serve as candidates for novel drug targets. TCSs consist of a sensor histidine kinase (HK) and its cognate response regulator (RR). Upon perception of a signal, HKs autophosphorylate their conserved histidine residues, followed by phosphotransfer to their partner RRs. The phosphorylated RRs mostly function as transcriptional regulators and control the expression of genes necessary for stress response. HKs sense their specific signals not only in their extracytoplasmic sensor domain but also in their cytoplasmic and transmembrane domains. The signals are sensed either directly or indirectly via cofactors and accessory proteins. Accumulating evidence shows that a single HK can sense and respond to multiple signals in different domains. The underlying molecular mechanisms of how HK activity is controlled by these signals have been extensively studied both biochemically and structurally. In this article, we introduce the wide diversity of signal perception in different domains of HKs, together with their recently clarified structures and molecular mechanisms.
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Project description:Sensor histidine kinases regulate adaptive cellular responses to changes in the chemical or physical state of the environment. HWE/HisKA2-family kinases comprise a subset of histidine kinases that is defined by unique sequence motifs in both the catalytic and non-catalytic regions. Recent crystal structures have defined conserved intramolecular interactions that inform models of kinase regulation that are unique to the HWE/HisKA2 superfamily. Emerging genetic, biochemical and genomic data indicate that, unlike typical histidine kinases, HWE/HisKA2 kinases do not generally signal via classical DNA-binding response regulators. Rather, these unusual kinases are often part of atypical regulatory pathways that control changes in gene expression via modulation of protein-protein interactions or transcription anti-termination.
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Project description:Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.
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Project description:Nitrate and nitrite transport across biological membranes is often facilitated by protein transporters that are members of the major facilitator superfamily. Paracoccus denitrificans contains an unusual arrangement whereby two of these transporters, NarK1 and NarK2, are fused into a single protein, NarK, which delivers nitrate to the respiratory nitrate reductase and transfers the product, nitrite, to the periplasm. Our complementation studies, using a mutant lacking the nitrate/proton symporter NasA from the assimilatory nitrate reductase pathway, support that NarK1 functions as a nitrate/proton symporter while NarK2 is a nitrate/nitrite antiporter. Through the same experimental system, we find that Escherichia coli NarK and NarU can complement deletions in both narK and nasA in P. denitrificans, suggesting that, while these proteins are most likely nitrate/nitrite antiporters, they can also act in the net uptake of nitrate. Finally, we argue that primary sequence analysis and structural modelling do not readily explain why NasA, NarK1 and NarK2, as well as other transporters from this protein family, have such different functions, ranging from net nitrate uptake to nitrate/nitrite exchange.
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Project description:Histidine kinases (HKs) are primary sensor proteins that act in cell signaling pathways generically referred to as "two-component systems" (TCSs). TCSs are among the most widely distributed transduction systems used by both prokaryotic and eukaryotic organisms to detect and respond to a broad range of environmental cues. The structure and distribution of HK proteins are now well documented in prokaryotes, but information is still fragmentary for eukaryotes. Here, we have taken advantage of recent genomic resources to explore the structural diversity and the phylogenetic distribution of HKs in the prominent eukaryotic supergroups. Searches of the genomes of 67 eukaryotic species spread evenly throughout the phylogenetic tree of life identified 748 predicted HK proteins. Independent phylogenetic analyses of predicted HK proteins were carried out for each of the major eukaryotic supergroups. This allowed most of the compiled sequences to be categorized into previously described HK groups. Beyond the phylogenetic analysis of eukaryotic HKs, this study revealed some interesting findings: 1) characterization of some previously undescribed eukaryotic HK groups with predicted functions putatively related to physiological traits; 2) discovery of HK groups that were previously believed to be restricted to a single kingdom in additional supergroups, and 3) indications that some evolutionary paths have led to the appearance, transfer, duplication, and loss of HK genes in some phylogenetic lineages. This study provides an unprecedented overview of the structure and distribution of HKs in the Eukaryota and represents a first step toward deciphering the evolution of TCS signaling in living organisms.
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Project description:NsaS is one of four intramembrane histidine kinases (HKs) in Staphylococcus aureus that mediate the pathogen's response to membrane active antimicrobials and human innate immunity. We describe the first integrative structural study of NsaS using a combination of solution state NMR spectroscopy, chemical-cross-linking, molecular modeling and dynamics. Three key structural features emerge: First, NsaS has a short N-terminal amphiphilic helix that anchors its transmembrane (TM) bundle into the inner leaflet of the membrane such that it might sense neighboring proteins or membrane deformations. Second, the transmembrane domain of NsaS is a 4-helix bundle with significant dynamics and structural deformations at the membrane interface. Third, the intracellular linker connecting the TM domain to the cytoplasmic catalytic domains of NsaS is a marginally stable helical dimer, with one state likely to be a coiled-coil. Data from chemical shifts, heteronuclear NOE, H/D exchange measurements and molecular modeling suggest that this linker might adopt different conformations during antibiotic induced signaling.
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Project description:Inorganic nitrate (NO3 - ), nitrite (NO2 - ) and NO are nitrogenous species with a diverse and interconnected chemical biology. The formation of NO from nitrate and nitrite via a reductive 'nitrate-nitrite-NO' pathway and resulting in vasodilation is now an established complementary route to traditional NOS-derived vasodilation. Nitrate, found in our diet and abundant in mammalian tissues and circulation, is activated via reduction to nitrite predominantly by our commensal oral microbiome. The subsequent in vivo reduction of nitrite, a stable vascular reserve of NO, is facilitated by a number of haem-containing and molybdenum-cofactor proteins. NO generation from nitrite is enhanced during physiological and pathological hypoxia and in disease states involving ischaemia-reperfusion injury. As such, modulation of these NO vascular repositories via exogenously supplied nitrite and nitrate has been evaluated as a therapeutic approach in a number of diseases. Ultimately, the chemical biology of nitrate and nitrite is governed by local concentrations, reaction equilibrium constants, and the generation of transient intermediates, with kinetic rate constants modulated at differing physiological pH values and oxygen tensions. LINKED ARTICLES: This article is part of a themed section on Nitric Oxide 20 Years from the 1998 Nobel Prize. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.2/issuetoc.
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Project description:The European Commission asked EFSA for a scientific opinion on the risks to animal health related to nitrite and nitrate in feed. For nitrate ion, the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) identified a BMDL 10 of 64 mg nitrate/kg body weight (bw) per day for adult cattle, based on methaemoglobin (MetHb) levels in animal's blood that would not induce clinical signs of hypoxia. The BMDL 10 is applicable to all bovines, except for pregnant cows in which reproductive effects were not clearly associated with MetHb formation. Since the data available suggested that ovines and caprines are not more sensitive than bovines, the BMDL 10 could also be applied to these species. Highest mean exposure estimates of 53 and 60 mg nitrate/kg bw per day in grass silage-based diets for beef cattle and fattening goats, respectively, may raise a health concern for ruminants when compared with the BMDL 10 of 64 mg nitrate/kg bw per day. The concern may be higher because other forages might contain higher levels of nitrate. Highest mean exposure estimates of 2.0 mg nitrate/kg bw per day in pigs' feeds indicate a low risk for adverse health effects, when compared with an identified no observed adverse effect level (NOAEL) of 410 mg nitrate/kg bw per day, although the levels of exposure might be underestimated due to the absence of data on certain key ingredients in the diets of this species. Due to the limitations of the data available, the CONTAM Panel could not characterise the health risk in species other than ruminants and pigs from nitrate and in all livestock and companion animals from nitrite. Based on a limited data set, both the transfer of nitrate and nitrite from feed to food products of animal origin and the nitrate- and nitrite-mediated formation of N-nitrosamines and their transfer into these products are likely to be negligible.
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Project description:NarK nitrate/nitrite antiporter imports nitrate (a mineral form of the essential element nitrogen) into the cell and exports nitrite (a metabolite that can be toxic in high concentrations) out of the cell. However, many details about its operational mechanism remain poorly understood. In this work, we performed steered molecular dynamics simulations of anion translocations and quantum-chemistry model calculations of the binding sites to study the wild-type NarK protein and its R89K mutant. Our results shed light on the importance of the two strictly conserved binding-site arginine residues (R89 and R305) and two glycine-rich signature motifs (G164-M176 and G408-F419) in anion movement through the pore. We also observe conformational changes of the protein during anion migration. For the R89K mutant, our quantum calculations reveal a competition for a proton between the anion (especially nitrite) and lysine, which can potentially slow down or even trap the anion in the pore. Our findings provide a possible explanation for the striking experimental finding that the arginine-to-lysine mutation, despite preserving the charge, impedes or abolishes anion transport in such mutants of NarK and other similar nitrate/nitrite exchangers.
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