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J Biol Chem. 2015 Jan 30;290(5):2630-43. doi: 10.1074/jbc.M114.618199. Epub 2014 Dec 5.

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.

Author information

1
From the Departments of Microbiology.
2
Chemistry, and.
3
Physics and Astronomy, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada and.
4
the Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
5
From the Departments of Microbiology, brian.mark@umanitoba.ca.

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.

KEYWORDS:

AmpC beta-Lactamase; AmpR; Crystal Structure; LysR-type Transcriptional Regulator; Mass Spectrometry (MS); Peptidoglycan; Small Angle X-ray Scattering (SAXS); Transcription

PMID:
25480792
PMCID:
PMC4316999
DOI:
10.1074/jbc.M114.618199
[Indexed for MEDLINE]
Free PMC Article

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