4WKM

AmpR effector binding domain from Citrobacter freundii bound to UDP-MurNAc-pentapeptide

Summary for 4WKM

• Entry DOI: 10.2210/pdb4wkm/pdb
• Related: 3kos 3kot
• Descriptor: LysR family transcriptional regulator, ALA-FGA-API-DAL-DAL, GLYCEROL, ... (5 entities in total)
• Functional Keywords: lysr-type transcriptional regulator, lttr, udp-murnac-pentapeptide, transcription
• Biological source: Citrobacter freundii ATCC 8090 = MTCC 1658; More
• Total number of polymer chains: 16
• Total formula weight: 204709.12

Authors

Vadlamani, G.,Reeve, T.M.,Mark, B.L. (deposition date: 2014-10-02, release date: 2014-12-17, Last modification date: 2023-11-15)

Primary citation

Vadlamani, G.,Thomas, M.D.,Patel, T.R.,Donald, L.J.,Reeve, T.M.,Stetefeld, J.,Standing, K.G.,Vocadlo, D.J.,Mark, B.L.
The beta-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.
J.Biol.Chem., 290:2630-2643, 2015

PubMed 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.

PubMed: 25480792
DOI: 10.1074/jbc.M114.618199

Experimental method

X-RAY DIFFRACTION (2.15 Å)