Journal: Structure / Year: 2010 Title: Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR. Authors: Goragot Wisedchaisri / David M Dranow / Thomas J Lie / Jeffrey B Bonanno / Yury Patskovsky / Sinem A Ozyurt / J Michael Sauder / Steven C Almo / Stephen R Wasserman / Stephen K Burley / John ...Authors: Goragot Wisedchaisri / David M Dranow / Thomas J Lie / Jeffrey B Bonanno / Yury Patskovsky / Sinem A Ozyurt / J Michael Sauder / Steven C Almo / Stephen R Wasserman / Stephen K Burley / John A Leigh / Tamir Gonen / Abstract: Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. ...Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.
History
Deposition
Aug 4, 2010
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Header (metadata) release
Nov 17, 2010
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Map release
Nov 17, 2010
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Update
Nov 17, 2010
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Current status
Nov 17, 2010
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
pH: 7.5 / Details: 100mM Tris HCl pH 7.5, 1M KCl and 5mM glycerol
Staining
Type: NEGATIVE Details: A 2 microlitre drop of NrpR was applied to a carbon-coated grid, washed 3 times with milliQ water and stained using 0.075% uranyl formate.
Vitrification
Cryogen name: NONE / Instrument: OTHER
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Electron microscopy
Microscope
FEI TECNAI 12
Image recording
Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000
Electron beam
Acceleration voltage: 120 kV / Electron source: LAB6
Specimen holder: Eucentric / Specimen holder model: OTHER
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Image processing
CTF correction
Details: Each particle
Final reconstruction
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider Details: Random conical tilt followed by angular refinement and reconstruction Number images used: 8200
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