Muropeptide binding and the X-ray structure of the effector domain of the transcriptional regulator AmpR of Pseudomonas aeruginosa

Abstract

A complex link exists between cell-wall recycling/repair and the manifestation of resistance to β-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of β-lactamase, an antibiotic-resistance determinant. The effector-binding domain (EBD) of AmpR was purified to homogeneity. We document that the EBD exists exclusively as a dimer, even at a concentration as low as 1 μM. The EBD binds to the suppressor ligand UDP-N-acetyl-β-D-muramyl-L-Ala-γ-D-Glu-meso-DAP-D-Ala-D-Ala and binds to two activator muropeptides, N-acetyl-β-D-glucosamine-(1→4)-1,6-anhydro-N-acetyl-β-D-muramyl-L-Ala-γ-D-Glu-meso-DAP-D-Ala-D-Ala and 1,6-anhydro-N-acetyl-β-D-muramyl-L-Ala-γ-D-Glu-meso-DAP-D-Ala-D-Ala, as assessed by non-denaturing mass spectrometry. The EBD does not bind to 1,6-anhydro-N-acetyl-β-D-muramyl-L-Ala-γ-D-Glu-meso-DAP. This binding selectivity revises the dogma in the field. The crystal structure of the EBD dimer was solved to 2.2 Å resolution. The EBD crystallizes in a >closed> conformation, in contrast to the >open> structure required to bind the muropeptides. Structural issues of this ligand recognition are addressed by molecular dynamics simulations, which reveal significant differences among the complexes with the effector molecules.