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AmpR effector binding domain from Citrobacter freundii bound to UDP-MurNAc-pentapeptideAmpR effector binding domain from Citrobacter freundii bound to UDP-MurNAc-pentapeptide
Structural highlights
Publication Abstract from PubMedInducible expression of chromosomal AmpC beta-lactamase is a major cause of beta-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 beta-lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-MurNAc-pentapeptide. When exposed to beta-lactams however, PG catabolites (1,6-anhydroMurNAc-peptides) accumulate in the cytosol, which have been proposed to competitively displace UDP-MurNAcpentapeptide 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-MurNAcpentapeptide. Non-denaturing mass spectrometry revealed AmpR to be a homotetramer that is stabilized by DNA containing the T-N11-A LTTR binding motif, and 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 supports previous claims that 1,6-anhydro-MurNAc-pentapeptide converts AmpR into an activator of ampC transcription more effectively than 1,6-anhydro-MurNAc-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-MurNAcpentapeptide predicts that the UDP-MurNAc moiety of the repressor participates in modulating AmpR function. The beta-lactamase Gene Regulator AmpR is a Tetramer that Recognizes and Binds the D-Ala-D-Ala Motif of its Repressor UDP-MurNAc-pentapeptide.,Vadlamani G, Thomas MD, Patel TR, Donald LJ, Reeve TM, Stetefeld J, Standing KG, Vocadlo DJ, Mark BL J Biol Chem. 2014 Dec 5. pii: jbc.M114.618199. PMID:25480792[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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