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A sensory adaptation system that tunes chemoreceptor sensitivity enables motile Escherichia coli cells to track chemical gradients with high sensitivity over a wide dynamic range. Sensory adaptation involves feedback control of covalent receptor modifications by two enzymes: CheR, a methyltransferase, and CheB, a methylesterase. This study describes a CheR function that opposes the signaling consequences of its catalytic activity. In the presence of CheR, a variety of mutant serine chemoreceptors displayed up to 40-fold enhanced detection sensitivity to chemoeffector stimuli. This response enhancement effect did not require the known catalytic activity of CheR, but did involve a binding interaction between CheR and receptor molecules. Response enhancement was maximal at low CheR:receptor stoichiometry and quantitative analyses argued against a reversible binding interaction that simply shifts the ON-OFF equilibrium of receptor signaling complexes. Rather, a short-lived CheR binding interaction appears to promote a long-lasting change in receptor molecules, either a covalent modification or conformation that enhances their response to attractant ligands.

Motile bacteria use large receptor arrays to detect chemical and physical stimuli in their environment, process this complex information, and accordingly bias their swimming in a direction they deem favorable. The chemoreceptor molecules form tripod-like trimers of receptor dimers through direct contacts between their cytoplasmic tips. A pair of trimers, together with a dedicated kinase enzyme, form a core signaling complex. Hundreds of core complexes network to form extended arrays. While considerable progress has been made in revealing the hierarchical structure of the array, the molecular properties underlying signal processing in these structures remain largely unclear. Here we analyzed the signaling properties of nonnetworked core complexes in live cells by following both conformational and kinase control responses to attractant stimuli and to output-biasing lesions at various locations in the receptor molecule. Contrary to the prevailing view that individual receptors are binary two-state devices, we demonstrate that conformational coupling between the ligand binding and the kinase-control receptor domains is, in fact, only moderate. In addition, we demonstrate communication between neighboring receptors through their trimer-contact domains that biases them to adopt similar signaling states. Taken together, these data suggest a view of signaling in receptor trimers that allows significant signal integration to occur within individual core complexes. [ABSTRACT FROM AUTHOR]

The chemotaxis machinery of Escherichia coli has served as a model for exploring the molecular signaling mechanisms of transmembrane chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). Yet, fundamental questions about signal transmission through MCP molecules remain unanswered. Our work with the E. coli serine chemoreceptor Tsr has developed in vivo reporters that distinguish kinase-OFF and kinase-ON structures in the cytoplasmic methylation helix (MH) cap, which receives stimulus signals from an adjoining, membrane-proximal histidine kinase, adenylyl cyclases, MCPs, and phosphatases (HAMP) domain. The cytoplasmic helices of the Tsr homodimer interact mainly through packing interactions of hydrophobic residues at a and d heptad positions. We investigated the in vivo crosslinking properties of Tsr molecules bearing cysteine replacements at functionally tolerant g heptad positions in the N-terminal and C-terminal cap helices. Upon treatment of cells with bismaleimido-ethane (BMOE), a bifunctional thiol-reagent, Tsr-G273C/Q504C readily formed a doubly crosslinked product in the presence of serine but not in its absence. Moreover, a serine stimulus combined with BMOE treatment during in vivo Förster resonance energy transfer–based kinase assays locked Tsr-G273C/Q504C in kinase-OFF output. An OFF-shifting lesion in MH1 (D269P) promoted the formation of the doubly crosslinked species in the absence of serine, whereas an ON-shifting lesion (G268P) suppressed the formation of the doubly crosslinked species. Tsr-G273C/Q504C also showed output-dependent crosslinking patterns in combination with ON-shifting and OFF-shifting adaptational modifications. Our results are consistent with a helix breathing-axial rotation-bundle repacking signaling mechanism and imply that in vivo crosslinking tools could serve to probe helix-packing transitions and their output consequences in other regions of the receptor molecule. [ABSTRACT FROM AUTHOR]

Motile Escherichia coli cells use chemoreceptor signaling arrays to track chemical gradients with exquisite precision. Highly conserved residues in the cytoplasmic hairpin tip of chemoreceptor molecules promote assembly of trimer-based signaling complexes and modulate the activity of their CheA kinase partners. To explore hairpin tip output states in the serine receptor Tsr, we characterized the signaling consequences of amino acid replacements at the salt-bridge residue pair E385-R388. All mutant receptors assembled trimers and signaling complexes, but most failed to support serine chemotaxis in soft agar assays. Small side-chain replacements at either residue produced OFF- or ON-shifted outputs that responded to serine stimuli in wild-type fashion, suggesting that these receptors, like the wild-type, operate as two-state signaling devices. Larger aliphatic or aromatic side chains caused slow or partial kinase control responses that proved dependent on the connections between core signaling units that promote array cooperativity. In a mutant lacking one of two key adapter-kinase contacts (interface 2), those mutant receptors exhibited more wild-type behaviors. Lastly, mutant receptors with charged amino acid replacements assembled signaling complexes that were locked in kinase-ON (E385K|R) or kinase-OFF (R388D|E) output. The hairpin tips of mutant receptors with these more aberrant signaling properties probably have nonnative structures or dynamic behaviors. Our results suggest that chemoeffector stimuli and adaptational modifications influence the cooperative connections between core signaling units. This array remodeling process may involve activity-dependent changes in the relative strengths of interface 1 and 2 interactions between the CheW and CheA.P5 components of receptor core signaling complexes. [ABSTRACT FROM AUTHOR]

A sensory adaptation system that tunes chemoreceptor sensitivity enables motile Escherichia coli cells to track chemical gradients with high sensitivity over a wide dynamic range. Sensory adaptation involves feedback control of covalent receptor modifications by two enzymes: CheR, a methyltransferase, and CheB, a methylesterase. This study describes a CheR function that opposes the signaling consequences of its catalytic activity. In the presence of CheR, a variety of mutant serine chemoreceptors displayed up to 40-fold enhanced detection sensitivity to chemoeffector stimuli. This response enhancement effect did not require the known catalytic activity of CheR, but did involve a binding interaction between CheR and receptor molecules. Response enhancement was maximal at low CheR:receptor stoichiometry and quantitative analyses argued against a reversible binding interaction that simply shifts the ON-OFF equilibrium of receptor signaling complexes. Rather, a short-lived CheR binding interaction appears to promote a long-lasting change in receptor molecules, either a covalent modification or conformation that enhances their response to attractant ligands. [ABSTRACT FROM AUTHOR]

Lipoxins and aspirin-triggered 15-epi-lipoxins (ATL) are counter- regulatory eicosanoids with potent antiinflammatory actions. Oral efficacy and mechanism of action of ZK-192, a β-oxidation-resistant 3-oxa-ATL analog, were examined in trinitrobenzenesulphonate (TNBS)-induced colitis. When dosed orally once daily, 300 and 1,000 µg/kg ZK-192 markedly attenuated TNBS colitis in rodents both in preventive and therapeutic regimens. ZK-192 attenuated weight loss, macroscopic and histologic colon injury, mucosal neutrophil infiltration, and colon wall thickening. ZK-192 was as effective as 3-10 mg/kg oral prednisolone. ZK-192 decreased mucosal mRNA levels for several inflammatory mediators: inducible nitric oxide synthase, cyclooxygenase 2, and macrophage inflammatory protein 2. ZK-192 also decreased mucosal mRNA and protein levels of T helper 1 effector cytokines: tumor necrosis factor α, IL-2, and IFN-α. Systemic levels of these cytokines were also dramatically attenuated. CD3/CD28-mediated costimulation of T helper 1 effector cytokine release in lamina propria mononuclear cells was markedly inhibited by ZK-192 ex vivo and in vitro. ZK-192 also prevented colitis in lymphocyte-deficient severe combined immunodeficient mice, with ≈ 75% inhibition of mucosal tumor necrosis factor α and IL-2 levels. The results are further evidence that innate immune cells function as triggers for hapten-induced colitis. The combined antiinflammatory and immunomodulatory effects of ZK-192 in TNBS colitis suggest that All analogs may be an attractive oral treatment approach for inflammatory bowel diseases. [ABSTRACT FROM AUTHOR]

Organometallic chemistry offers novel concepts in structural diversity and molecular recognition that can be used in drug design. Here, we consider DNA recognition by n[sup6]-arene Ru(ll) anticancer complexes by an induced-fit mechanism. The stereochemistry of the dinuclear complex [((n[sup6]-biphenyl)RuCl(en))[sub2](CH[sub2])[sub6]][sup2+] (3, en = ethylenediamine) was elucidated by studies of the half unit [(n[sup6]-biphenyl)RuCl(Et-en)]+ (2, where Et-en is Et(H)NCH[sub2]CH[sub2]NH[sub2]). The structures of the separated R[sup+][subRu]R[sup+][subN] and S[sup+][subRu]R[sup+][subN] diastereomers of 2 were determined by x-ray crystallography; their slow interconversion in water (t½ ≈ 2 h, 298 K, pH 6.2) was observed by NMR spectroscopy. For 2 and 3 the R[sup+][subRu]R[sup+][subN] configurations are more stable than S[sup+][subRu]R[sup+][subN] (73:27). X-ray and NMR studies showed that reactions of 2 and 3 with 9-ethylguanine gave rise selectively to S[sup+][subRu]R[sup+][subN] diastereomers. Dynamic chiral recognition of guanine can lead to high diastereoselectivity of DNA binding. The dinuclear complex 3 induced a large unwinding (31°) of plasmid DNA, twice that of mononuclear 2 (14°), and effectively inhibited DNA-directed RNA synthesis in vitro. This dinuclear complex gave rise to interstrand cross-links on a 213-bp plasmid fragment with efficiency similar to bifunctional cisplatin, and to 1,3-GG interstrand and 1,2-GG and 1,3-GTG intrastrand cross-links on site-specifically ruthenated 20-mers. Complex 3 blocked intercalation of ethidium considerably more than mononuclear 2. The concept of induced-fit recognition of DNA by organometallic complexes containing dynamic stereogenic centers via dynamic epimerization, intercalation, and cross-linking may be useful in the design of anticancer drugs. [ABSTRACT FROM AUTHOR]

Motile Escherichia coli cells use chemoreceptor signaling arrays to track chemical gradients with exquisite precision. Highly conserved residues in the cytoplasmic hairpin tip of chemoreceptor molecules promote assembly of trimer-based signaling complexes and modulate the activity of their CheA kinase partners. To explore hairpin tip output states in the serine receptor Tsr, we characterized the signaling consequences of amino acid replacements at the salt-bridge residue pair E385-R388. All mutant receptors assembled trimers and signaling complexes, but most failed to support serine chemotaxis in soft agar assays. Small side-chain replacements at either residue produced OFF- or ON-shifted outputs that responded to serine stimuli in wild-type fashion, suggesting that these receptors, like the wild-type, operate as two-state signaling devices. Larger aliphatic or aromatic side chains caused slow or partial kinase control responses that proved dependent on the connections between core signaling units that promote array cooperativity. In a mutant lacking one of two key adapter-kinase contacts (interface 2), those mutant receptors exhibited more wild-type behaviors. Lastly, mutant receptors with charged amino acid replacements assembled signaling complexes that were locked in kinase-ON (E385K|R) or kinase-OFF (R388D|E) output. The hairpin tips of mutant receptors with these more aberrant signaling properties probably have nonnative structures or dynamic behaviors. Our results suggest that chemoeffector stimuli and adaptational modifications influence the cooperative connections between core signaling units. This array remodeling process may involve activity-dependent changes in the relative strengths of interface 1 and 2 interactions between the CheW and CheA.P5 components of receptor core signaling complexes., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Lipoxins and aspirin-triggered 15-epi-lipoxins (ATL) are counter-regulatory eicosanoids with potent antiinflammatory actions. Oral efficacy and mechanism of action of ZK-192, a beta-oxidation-resistant 3-oxa-ATL analog, were examined in trinitrobenzenesulphonate (TNBS)-induced colitis. When dosed orally once daily, 300 and 1,000 mug/kg ZK-192 markedly attenuated TNBS colitis in rodents both in preventive and therapeutic regimens. ZK-192 attenuated weight loss, macroscopic and histologic colon injury, mucosal neutrophil infiltration, and colon wall thickening. ZK-192 was as effective as 3-10 mg/kg oral prednisolone. ZK-192 decreased mucosal mRNA levels for several inflammatory mediators: inducible nitric oxide synthase, cyclooxygenase 2, and macrophage inflammatory protein 2. ZK-192 also decreased mucosal mRNA and protein levels of T helper 1 effector cytokines: tumor necrosis factor alpha, IL-2, and IFN-gamma. Systemic levels of these cytokines were also dramatically attenuated. CD3/CD28-mediated costimulation of T helper 1 effector cytokine release in lamina propria mononuclear cells was markedly inhibited by ZK-192 ex vivo and in vitro. ZK-192 also prevented colitis in lymphocyte-deficient severe combined immunodeficient mice, with approximately 75% inhibition of mucosal tumor necrosis factor alpha and IL-2 levels. The results are further evidence that innate immune cells function as triggers for hapten-induced colitis. The combined antiinflammatory and immunomodulatory effects of ZK-192 in TNBS colitis suggest that ATL analogs may be an attractive oral treatment approach for inflammatory bowel diseases.