Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR.
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.
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Goragot
G
Wisedchaisri
David M
DM
Dranow
Thomas J
TJ
Lie
Jeffrey B
JB
Bonanno
Yury
Y
Patskovsky
Sinem A
SA
Ozyurt
J Michael
JM
Sauder
Steven C
SC
Almo
Stephen R
SR
Wasserman
Stephen K
SK
Burley
John A
JA
Leigh
Tamir
T
Gonen
D019848:Gene Expression Regulation, Archaeal / Q000235:genetics*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D017017:Methanococcus / Q000737:chemistry*; D008854:Microscopy, Electron; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009584:Nitrogen / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D007656:Ketoglutaric Acids; D051922:PII Nitrogen Regulatory Proteins; D000644:Quaternary Ammonium Compounds; D014157:Transcription Factors; D009584:Nitrogen