title: The β-lactamase gene regulator AmpR is a tetramer that recognizes and binds the D-Ala-D-Ala motif of its repressor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide.
abstract: Inducible expression of chromosomal AmpC β-lactamase is a major cause of β-lactam antibiotic resistance in the Gram-negative bacteria Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is induced by the LysR-type transcriptional regulator (LTTR) AmpR, which activates ampC expression in response to changes in peptidoglycan (PG) metabolite levels that occur during exposure to β-lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide. When exposed to β-lactams, however, PG catabolites (1,6-anhydroMurNAc-peptides) accumulate in the cytosol, which have been proposed to competitively displace UDP-MurNAc-pentapeptide from AmpR and convert it into an activator of ampC transcription. Here we describe the molecular interactions between AmpR (from Citrobacter freundii), its DNA operator, and repressor UDP-MurNAc-pentapeptide. Non-denaturing mass spectrometry revealed AmpR to be a homotetramer that is stabilized by DNA containing the T-N11-A LTTR binding motif and revealed that it can bind four repressor molecules in an apparently stepwise manner. A crystal structure of the AmpR effector-binding domain bound to UDP-MurNAc-pentapeptide revealed that the terminal D-Ala-D-Ala motif of the repressor forms the primary contacts with the protein. This observation suggests that 1,6-anhydroMurNAc-pentapeptide may convert AmpR into an activator of ampC transcription more effectively than 1,6-anhydroMurNAc-tripeptide (which lacks the D-Ala-D-Ala motif). Finally, small angle x-ray scattering demonstrates that the AmpR·DNA complex adopts a flat conformation similar to the LTTR protein AphB and undergoes only a slight conformational change when binding UDP-MurNAc-pentapeptide. Modeling the AmpR·DNA tetramer bound to UDP-MurNAc-pentapeptide predicts that the UDP-MurNAc moiety of the repressor participates in modulating AmpR function.
authors.affiliation: From the Departments of Microbiology.
authors.forename: Grishma
authors.initials: G
authors.lastname: Vadlamani
authors.affiliation: From the Departments of Microbiology.
authors.forename: Misty D
authors.initials: MD
authors.lastname: Thomas
authors.affiliation: Chemistry, and.
authors.forename: Trushar R
authors.initials: TR
authors.lastname: Patel
authors.affiliation: Chemistry, and.
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Donald
authors.affiliation: From the Departments of Microbiology.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Reeve
authors.affiliation: Chemistry, and.
authors.forename: Jörg
authors.initials: J
authors.lastname: Stetefeld
authors.affiliation: Physics and Astronomy, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada and.
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Standing
authors.affiliation: the Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
authors.affiliation: From the Departments of Microbiology, brian.mark@umanitoba.ca.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry* / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D011485:Protein Binding; D053838:Scattering, Small Angle; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C071526:AmpR protein, Bacteria; C002225:UDP-N-acetylmuramic acid pentapeptide; C002956:alanylalanine; C048795:AmpC beta-lactamases; D001618:beta-Lactamases
keywords: AmpC beta-Lactamase; AmpR; Crystal Structure; LysR-type Transcriptional Regulator; Mass Spectrometry (MS); Peptidoglycan; Small Angle X-ray Scattering (SAXS); Transcription