NMR structure of activated CheY

Cho, Ho S.; Lee, Seok Yong; Yan, Dalai; Pan, Xiaoyu; Parkinson, John S.; Kustu, Sydney; Wemmer, David E.; Pelton, Jeffrey G.

doi:10.1006/jmbi.2000.3595

Cited by 131 publications

(198 citation statements)

References 54 publications

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“…This implies that the position of the switches alone is not sufficient to define the states and a kinetic definition of the active and inactive states (described below) must be used for understanding the activation mechanism. The probability density distributions also provide evidence against the commonly accepted theory related to the activation of response regulators such as NtrC and CheY 21 . In two-component systems like these, activation of the response regulator receiver domains has been proposed to rely on a 'Y-T coupling' mechanism 22 (Y101 and T82 in NtrC), where the interaction between this conserved pair of residues is correlated with the activation of the protein.…”

Section: Resultssupporting

confidence: 52%

A network of molecular switches controls the activation of the two-component response regulator NtrC

Vanatta

Shukla²,

Lawrenz

et al. 2015

Nat Commun

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Recent successes in simulating protein structure and folding dynamics have demonstrated the power of molecular dynamics to predict the long timescale behaviour of proteins. Here, we extend and improve these methods to predict molecular switches that characterize conformational change pathways between the active and inactive state of nitrogen regulatory protein C (NtrC). By employing unbiased Markov state model-based molecular dynamics simulations, we construct a dynamic picture of the activation pathways of this key bacterial signalling protein that is consistent with experimental observations and predicts new mutants that could be used for validation of the mechanism. Moreover, these results suggest a novel mechanistic paradigm for conformational switching.

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Section: Resultssupporting

confidence: 52%

A network of molecular switches controls the activation of the two-component response regulator NtrC

Vanatta

Shukla²,

Lawrenz

et al. 2015

Nat Commun

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“…To assess quantitatively whether the determinants of NepR recognition indeed reside exclusively in PhyR SL , the affinities of NepR for activated PhyR and for the isolated PhyR SL domain were compared using isothermal titration calorimetry (ITC). The phosphoryl analog beryllium fluoride (BeF 3 ) (18,25,26) was used to activate PhyR. PhyR-BeF 3 and PhyR SL were found to bind NepR in a 1:1 stoichiometry with dissociation constants of 31.3 ± 2.7 nM and 5.6 ± 1.0 nM, respectively ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, the surface of PhyR that binds NepR helix α1′ in the PhyR SL -NepR complex interacts in unphosphorylated PhyR with the α11-β5-α12 surface of the receiver domain (Fig. 3B), the region known to undergo an allosteric transition on receiver domain phosphorylation (17)(18)(19). To test whether contacts in the α11-β5-α12 region of PhyR REC are major determinants of the inhibitory interactions in PhyR, residue E235 in the α11-β5 loop of PhyR REC , which makes polar contacts with the side chains of residues R19 and Q25 in the PhyR SL domain, was substituted with alanine in a nonphosphorylatable PhyR allele, PhyR D194A (20).…”

Section: Structural Comparison Between the Phyr Sl -Nepr Complex Andmentioning

confidence: 99%

“…The crystal structure of the unphosphorylated form of PhyR from Caulobacter crescentus shows that the two domains of PhyR, PhyR REC and PhyR SL , interact through a polar interface that buries one face of the PhyR SL domain and the α4-β5-α5 face of the receiver domain (16), a region known to undergo conformational changes on phosphorylation of the receiver domain in other response regulators (17)(18)(19). It was proposed that the activation of PhyR releases the PhyR SL domain to bind the antisigma factor NepR (12); however, the mode of binding of NepR to PhyR SL remains unknown.…”

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confidence: 99%

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Structural basis for sigma factor mimicry in the general stress response of Alphaproteobacteria

Campagne

Damberger

Kaczmarczyk³

et al. 2012

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

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Reprogramming gene expression is an essential component of adaptation to changing environmental conditions. In bacteria, a widespread mechanism involves alternative sigma factors that redirect transcription toward specific regulons. The activity of sigma factors is often regulated through sequestration by cognate anti-sigma factors; however, for most systems, it is not known how the activity of the anti-sigma factor is controlled to release the sigma factor. Recently, the general stress response sigma factor in Alphaproteobacteria, σ EcfG , was identified. σ EcfG is inactivated by the anti-sigma factor NepR, which is itself regulated by the response regulator PhyR. This key regulator sequesters NepR upon phosphorylation of its PhyR receiver domain via its σ EcfG sigma factor-like output domain (PhyR SL ). To understand the molecular basis of the PhyR-mediated partner-switching mechanism, we solved the structure of the PhyR SL –NepR complex using NMR. The complex reveals an unprecedented anti-sigma factor binding mode: upon PhyR SL binding, NepR forms two helices that extend over the surface of the PhyR SL subdomains. Homology modeling and comparative analysis of NepR, PhyR SL , and σ EcfG mutants indicate that NepR contacts both proteins with the same determinants, showing sigma factor mimicry at the atomic level. A lower density of hydrophobic interactions, together with the absence of specific polar contacts in the σ EcfG –NepR complex model, is consistent with the higher affinity of NepR for PhyR compared with σ EcfG . Finally, by reconstituting the partner switch in vitro, we demonstrate that the difference in affinity of NepR for its partners is sufficient for the switch to occur.

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“…This domain is 38 % identical to the same domain in E. coli CheY. The crystallographic structure of CheY reveals the presence of five highly conserved residues in the response regulator active site: Asp57 (the site of phosphorylation) (Sanders et al, 1989); Asp12 and Asp13 (involved in coordination of a Mg 2+ ion essential for phosphorylation and dephosphorylation) (Stock et al, 1993); Lys109 (important for the phosphorylation-induced conformational change) (Lukat et al, 1991); and Thr87 (also involved in conformational change through formation of a hydrogen bond between its OH group and the active site acceptor) (Appleby & Bourret, 1998;Cho et al, 2000). The corresponding sites in RcsC are Asp875, Asp831, Asp832, Lys925 and Thr903.…”

Section: Discussionmentioning

confidence: 99%

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

et al. 2005

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Mutations in rcsC that result in constitutive colanic acid capsule synthesis were obtained in Salmonella enterica serovar Typhimurium. Most rcsC alleles were dominant; however, recessive rcsC alleles were also found, in agreement with the postulated double role (positive and negative) of RcsC on the activation of the RcsB/C phosphorelay system. Salmonella rcsC mutants with constitutive activation of the Rcs system are severely attenuated for virulence in BALB/c mice and their degree of attenuation correlates with the level of Rcs activation. Partial relief of attenuation by a gmm mutation indicates that capsule overproduction is one of the factors leading to avirulence in constitutively activated rcsC mutants. INTRODUCTIONThe Rcs phosphorelay system was initially characterized in Escherichia coli as a two-component positive regulator of colanic acid capsule synthesis encoded by rcsC and rcsB (Brill et al., 1988;Gottesman et al., 1985;Stout & Gottesman, 1990). The sensor protein RcsC, a hybrid histidine kinase, and the transcriptional activator RcsB are the main components of the system, which also includes a second transcriptional activator, RcsA (Stout et al., 1991) and an intermediate phosphotransmitter, YojN (Takeda et al., 2001). The Rcs system is also a negative regulator of the flagellar master operon flhDC (Francez-Charlot et al., 2003), whose products are necessary for the expression of genes involved in flagellum synthesis, motility and chemotaxis.The first environmental signal shown to strongly and rapidly induce colanic acid synthesis was osmotic upshift (Sledjeski & Gottesman, 1996). This effect was mediated by the Rcs system. More recently it has been shown that the Rcs phosphorelay can be activated when wild-type cells are grown at 20 uC in the presence of glucose and a high concentration of Zn 2+ (Hagiwara et al., 2003). Furthermore, several genetic conditions that result in alterations in envelope composition or integrity are known to activate the Rcs system. For example, a mutation in the mdoH gene involved in the biosynthesis of membrane-derived oligosaccharides (Ebel et al., 1997) Here, we describe the isolation of Salmonella rcsC mutants with constitutive activation of the Rcs system, and the characterization of amino acid substitutions that lead to different degrees of activation of the system. Virulence trials with constitutively activated rcsC mutants provide a direct demonstration of the link between activation of the Rcs system and avirulence in mice. We also show that virulence attenuation associated with activation of the RcsB/C system is partially due to colanic acid overproduction. METHODSBacterial strains, bacteriophages and strain construction.S. enterica serovar Typhimurium strains used in this study are described in Table 1. Unless otherwise indicated, the strains derive from the mouse-virulent strain 14028. Transductional crosses using phage P22 HT 105/1 int201 (Schmieger, 1972) were used for strain construction operations. The transduction protocol was described by Maloy (199...

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NMR structure of activated CheY

Cited by 131 publications

References 54 publications

A network of molecular switches controls the activation of the two-component response regulator NtrC

A network of molecular switches controls the activation of the two-component response regulator NtrC

Structural basis for sigma factor mimicry in the general stress response of Alphaproteobacteria

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

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