Improved radiation dose efficiency in solution SAXS using a sheath flow sample environment

Kirby, Nigel; Cowieson, Nathan; Hawley, Adrian; Mudie, Stephen; McGillivray, Duncan J.; Kusel, Michael; Samardzic-Boban, Vesna; Ryan, Timothy M.

doi:10.1107/s2059798316017174

Cited by 153 publications

(129 citation statements)

References 19 publications

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“…All data were analyzed using UltraScan 4.0, release 2578. 36 Sedimentation data were evaluated by the two-dimensional spectrum analysis (2DSA), 37 39 Scattering data was collected in 1 second exposures (λ = 1.0332 Å) over a total of 400 frames, using a 1.5 mm glass capillary, at 12 C. 2D intensity plots were radially averaged, normalized to sample transmission, and background subtracted using the scatterBrain software package (Australian Synchrotron).…”

Section: Analytical Ultracentrifugationmentioning

confidence: 99%

On the structure and function of Escherichia coli YjhC: An oxidoreductase involved in bacterial sialic acid metabolism

et al. 2019

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Human pathogenic and commensal bacteria have evolved the ability to scavenge host‐derived sialic acids and subsequently degrade them as a source of nutrition. Expression of the Escherichia coli yjhBC operon is controlled by the repressor protein nanR, which regulates the core machinery responsible for the import and catabolic processing of sialic acid. The role of the yjhBC encoded proteins is not known—here, we demonstrate that the enzyme YjhC is an oxidoreductase/dehydrogenase involved in bacterial sialic acid degradation. First, we demonstrate in vivo using knockout experiments that YjhC is broadly involved in carbohydrate metabolism, including that of N‐acetyl‐d‐glucosamine, N‐acetyl‐d‐galactosamine and N‐acetylneuraminic acid. Differential scanning fluorimetry demonstrates that YjhC binds N‐acetylneuraminic acid and its lactone variant, along with NAD(H), which is consistent with its role as an oxidoreductase. Next, we solved the crystal structure of YjhC in complex with the NAD(H) cofactor to 1.35 Å resolution. The protein fold belongs to the Gfo/Idh/MocA protein family. The dimeric assembly observed in the crystal form is confirmed through solution studies. Ensemble refinement reveals a flexible loop region that may play a key role during catalysis, providing essential contacts to stabilize the substrate—a unique feature to YjhC among closely related structures. Guided by the structure, in silico docking experiments support the binding of sialic acid and several common derivatives in the binding pocket, which has an overall positive charge distribution. Taken together, our results verify the role of YjhC as a bona fide oxidoreductase/dehydrogenase and provide the first evidence to support its involvement in sialic acid metabolism.

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Section: Analytical Ultracentrifugationmentioning

confidence: 99%

On the structure and function of Escherichia coli YjhC: An oxidoreductase involved in bacterial sialic acid metabolism

et al. 2019

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“…Here 50 μL of purified recombinant ASK1(269-658) at 7.7 mg/mL, ASK1(88-658) at 9.7 mg/mL, or ASK1(88-941) at 9.3 mg/mL (WT) or 11.5 mg/mL (W476S), were injected onto an inline Superdex 200 5/150 column (GE Healthcare) and eluted at a flow rate of 0.2 mL/min via a 1.5-mm glass capillary positioned in the X-ray beam in 500 mM NaCl, 10 mM Hepes (pH 7.5), 5% (vol/vol) glycerol, and 0.2 mM TCEP at 12°C. Coflow SAXS was used to minimize sample dilution and maximize signal to noise (72).…”

Section: Methodsmentioning

confidence: 99%

Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1

Weijman

Kumar

Jamieson

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.itogen-activated protein (MAP) kinase cascades transmit signals from membrane-associated receptors to intracellular targets to effect changes in cellular behavior. They form a hierarchical system in which activated upstream kinases (MAP3Ks) phosphorylate intermediate MAP kinase kinases (MAP2Ks), which in turn phosphorylate terminal MAP kinases, primarily ERK, p38, and JNK and their isoforms (1). Extensive studies have focused on the activation of RAS-RAF-MEK upstream in the ERK pathway and provided fertile ground for the discovery of new therapeutics (2). In contrast to the ERK pathway, which primarily promotes cellular proliferation, JNK and p38 phosphorylate a range of substrates to promote inflammation and cell death (1, 3). In addition, cross-regulation among the p38, JNK, and ERK pathways is important for the efficacy of various cancer therapies that are in use or in development (4, 5). Molecular details on the more diverse upstream regulation of the p38 and JNK pathways are currently less clear, however.Apoptosis signal-regulating kinases (ASK1-3) are MAP3Ks that trigger cellular responses to redox stress and inflammatory cytokines (6, 7) and play vital roles in innate immunity and viral infection (8-11). When activated, ASK1-3 activate JNK and p38 via phosphorylation of MAP2Ks (MKK3/4/6/7) (12). The key initiator role of ASK1-3 in this pathway means that either too much or too little ASK activity can have pathological effects. For instance, inhibiting ASK1 is beneficial against gastric cancer (13,14), but inactivating mutations in ASK1...

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“…SAXS data were collected at the Australian Synchrotron SAXS/WAXS beamline using an inline coflow size-exclusion chromatography setup to minimize sample dilution and maximize signal-to-noise 70 . Purified NanR at 10 mg mL -1 (340 µM) was injected (70 µL) onto an inline Superdex S200 5/150 Increase (GE Healthcare), equilibrated with buffer C, and supplemented with the radical scavenger 0.1% (w/v) sodium azide, using a flow rate of 0.45 mL min -1 .…”

Section: Small-angle X-ray Scattering (Saxs) Analysismentioning

confidence: 99%

Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

Horne¹,

Venugoapl

Panjikar

et al. 2020

Preprint

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Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Truncation of an N-terminal extension abolishes cooperative binding. The effector, N-acetylneuraminate, binds NanR and attenuates DNA binding. Crystal structure data show that N-acetylneuraminate binding to NanR causes a domain rearrangement that locks the protein in a conformation that prevents DNA binding. Single-particle cryo-electron microscopy structures of NanR bound to DNA reveal the DNA binding domain is reorganized to engage DNA, while the three dimers assemble in close proximity across the (GGTATA)3-repeat operator allowing protein-protein interactions to form via the Nterminal extensions. Our data provides a molecular basis for the regulation of bacterial sialic acid metabolism.Bacteria rapidly adapt to changes in nutrient availability. The physiological response to these changes is multi-layered, but a key element is gene regulation 1-3 . Genes that encode the appropriate metabolic machinery are induced, while those that are unnecessary are repressed.For example, the human gastrointestinal tract is heavily populated with bacteria 4-7 , but nutrient availability fluctuates 7-9 and glucose is often limiting 10-12 . Bacteria evolved the capacity to import and metabolize sialic acids, a diverse family of negatively-charged, nine-carbon amino monosaccharides [13][14][15] . Sialic acids coat host cell surfaces and are abundant in the mucosal epithelia of the gastrointestinal tract, where they mediate a variety of physiological and pathological processes 16 . Sialic acids are also a source of carbon, nitrogen, and energy for pathogenic and commensal bacteria 17,18 and some species also incorporate these sugars onto their cell surface to evade the human innate immune response 14,19,20 . Bacterial sialic acid metabolism is largely confined to mammalian commensal or pathogenic bacteria and most of these species colonize sialic acid-rich areas 17,18 , suggesting a link between sialic acid utilization and survival in the host.In Escherichia coli, the Nan repressor (NanR) regulates the expression of proteins responsible for sialic acid uptake and metabolism 21 (Fig. 1a). As a transcriptional repressor, NanR binds to a DNA operator site containing three GGTATA repeats 21,22 located within the promoter region of target genes and downstream of the RNA polymerase binding site thereby blocking transcription 18 . The GGTATA repeat operator is found in three operons (Fig. 1a-c) collectively referred to as the sialoregulon. This operon arrangement has been identified in E. coli, including Shiga toxin-producing strains, and Shigella dysenteriae 18,22 .E. coli NanR belongs to t...

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Improved radiation dose efficiency in solution SAXS using a sheath flow sample environment

Cited by 153 publications

References 19 publications

On the structure and function of Escherichia coli YjhC: An oxidoreductase involved in bacterial sialic acid metabolism

On the structure and function of Escherichia coli YjhC: An oxidoreductase involved in bacterial sialic acid metabolism

Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1

Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

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