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Evidence against the "Y-T coupling" mechanism of activation in the response regulator NtrC.

ABSTRACT: The dominant theory on the mechanism of response regulators activation in two-component bacterial signaling systems is the "Y-T coupling" mechanism, wherein the ?1 rotameric state of a highly conserved aromatic residue correlates with the activation of the protein via structural rearrangements coupled to a conserved tyrosine. In this paper, we present evidence that, in the receiver domain of the response regulator nitrogen regulatory protein C (NtrC(R)), the interconversion of this tyrosine (Y101) between its rotameric states is actually faster than the rate of inactive/active conversion and is not correlated to the activation process. Data gathered from NMR relaxation dispersion experiments show that a subset of residues surrounding the conserved tyrosine sense a process that is occurring at a faster rate than the inactive/active conformational transition. We show that this process is related to ?1 rotamer exchange of Y101 and that mutation of this aromatic residue to a leucine eliminated this second faster process without affecting activation. Computational simulations of NtrC(R) in its active conformation further demonstrate that the rotameric state of Y101 is uncorrelated with the global conformational transition during activation. Moreover, the tyrosine does not appear to be involved in the stabilization of the active form upon phosphorylation and is not essential in propagating the signal downstream for ATPase activity of the central domain. Our data provide experimental evidence against the generally accepted "Y-T coupling" mechanism of activation in NtrC(R).

SUBMITTER: Villali J

PROVIDER: S-EPMC4384162 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Evidence against the "Y-T coupling" mechanism of activation in the response regulator NtrC.

Villali Janice J Pontiggia Francesco F Clarkson Michael W MW Hagan Michael F MF Kern Dorothee D

Journal of molecular biology 20140107 7

The dominant theory on the mechanism of response regulators activation in two-component bacterial signaling systems is the "Y-T coupling" mechanism, wherein the χ1 rotameric state of a highly conserved aromatic residue correlates with the activation of the protein via structural rearrangements coupled to a conserved tyrosine. In this paper, we present evidence that, in the receiver domain of the response regulator nitrogen regulatory protein C (NtrC(R)), the interconversion of this tyrosine (Y10 ...[more]

PMID: 24406745

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Project description:UnlabelledNitrogen regulation in Escherichia coli is a model system for gene regulation in bacteria. Growth on glutamine as a sole nitrogen source is assumed to be nitrogen limiting, inferred from slow growth and strong NtrB/NtrC-dependent gene activation. However, we show that under these conditions, the intracellular glutamine concentration is not limiting but 5.6-fold higher than in ammonium-replete conditions; in addition, α-ketoglutarate concentrations are elevated. We address this glutamine paradox from a systems perspective. We show that the dominant role of NtrC is to regulate glnA transcription and its own expression, indicating that the glutamine paradox is not due to NtrC-independent gene regulation. The absolute intracellular NtrC and GS concentrations reveal molecular control parameters, where NtrC-specific activities were highest in nitrogen-starved cells, while under glutamine growth, NtrC showed intermediate specific activity. We propose an in vivo model in which α-ketoglutarate can derepress nitrogen regulation despite nitrogen sufficiency.ImportanceNitrogen is the most important nutrient for cell growth after carbon, and its metabolism is coordinated at the metabolic, transcriptional, and protein levels. We show that growth on glutamine as a sole nitrogen source, commonly assumed to be nitrogen limiting and used as such as a model system for nitrogen limitation, is in fact nitrogen replete. Our integrative quantitative analysis of key molecules involved in nitrogen assimilation and regulation reveal that glutamine is not necessarily the dominant molecule signaling nitrogen sufficiency and that α-ketoglutarate may play a more important role in signaling nitrogen status. NtrB/NtrC integrates α-ketoglutarate and glutamine signaling--sensed by the UTase (glnD) and PII (glnB), respectively--and regulates the nitrogen response through self-regulated expression and phosphorylation-dependent activation of the nitrogen (ntr) regulon. Our findings support α-ketoglutarate acting as a global regulatory metabolite.

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Evidence against the "Y-T coupling" mechanism of activation in the response regulator NtrC.

Publications

Evidence against the "Y-T coupling" mechanism of activation in the response regulator NtrC.

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