title: The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis.
abstract: TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated L-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.
authors.affiliation: From the Interfaculty Institute for Microbiology and Infection Medicine, University of Tuebingen, Auf der Morgenstelle 28, 72076 Tuebingen, Germany.
authors.forename: Ksenia
authors.initials: K
authors.lastname: Hauf
authors.affiliation: the Department of Genetics, Kazan Federal University, Kremlevskaya 18, 420008, Kazan, Russia, and.
authors.forename: Airat
authors.initials: A
authors.lastname: Kayumov
authors.affiliation: Wyatt Technology Europe, Hochstrasse 12a, 56307 Dernbach, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Gloge
authors.affiliation: From the Interfaculty Institute for Microbiology and Infection Medicine, University of Tuebingen, Auf der Morgenstelle 28, 72076 Tuebingen, Germany, karl.forchhammer@uni-tuebingen.de.
authors.forename: Karl
authors.initials: K
authors.lastname: Forchhammer
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology / Q000378:metabolism*; D001426:Bacterial Proteins / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D004795:Enzyme Stability; D017353:Gene Deletion; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D012097:Repressor Proteins; C101458:ScgR protein, Bacillus subtilis; D005973:Glutamine; C054437:methionine sulfoximine phosphate; D008717:Methionine Sulfoximine; D018698:Glutamic Acid; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase
keywords: AMP; ATP; Bacillus subtilis; glutamate; glutamine; glutamine synthetase; transcription factor TnrA; transcription regulation