Abstract
Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC) recognizes tandem imperfect repeat sequences on the promoters of genes to induce antimicrobial peptide resistance after phosphorylation and dimerization of its N-terminal receiver domain (PmrAN). However, structural information concerning how phosphorylation of the response regulator enhances DNA recognition remains elusive. To gain insights, we determined the nuclear magnetic resonance solution structure of PmrAC and characterized the interactions between PmrAC or BeF3−-activated full-length PmrA (PmrAF) and two DNA sequences from the pbgP promoter of K. pneumoniae. We showed that PmrAC binds to the PmrA box, which was verified to contain two half-sites, 5′-CTTAAT-3′ and 5′-CCTAAG-3′, in a head-to-tail fashion with much stronger affinity to the first than the second site without cooperativity. The structural basis for the PmrAC–DNA complex was investigated using HADDOCK docking and confirmed by paramagnetic relaxation enhancement. Unlike PmrAC, PmrAF recognizes the two sites simultaneously and specifically. In the PmrAF–DNA complex, PmrAN may maintain an activated homodimeric conformation analogous to that in the free form and the interactions between two PmrAC molecules aid in bending and binding of the DNA duplex for transcription activation.
Highlights
Bacteria are highly adaptive organisms whose genomes harbor many genes and pathways for sensing and responding to environmental cues
Based on the experimentally verified PmrA boxes in Salmonella typhimurium (In Supplementary Figure S1, K. pneumoniae PmrA shares 91% sequence identity to Salmonella PmrA for the C-terminal region Leu151 to Leu216), only CYT5 to THY10 and CYT16 to GUA21 were selected as active bases
DNase footprinting experiments with the PmrA protein from Salmonella enterica (In Supplementary Figure S1, K. pneumoniae PmrA shares 91% sequence identity to Salmonella PmrA for the C-terminal region Leu151 to Leu216) demonstrated specific binding to the pbgP promoter at the predicted PmrA box (TCTTAAT ATTATCCTAAGC, half1 and half2 sites underlined) [38]
Summary
Bacteria are highly adaptive organisms whose genomes harbor many genes and pathways for sensing and responding to environmental cues. The two-component system (TCS) is one of the major ways of coupling environmental stimuli to adaptive responses [1]. A classical TCS typically consists of a transmembrane sensor histidine kinase (HK) and a cytoplasmic response regulator (RR) protein. After perceiving external stimuli by the sensor domain of the HK, a phosphoryl group on a highly conserved His residue of the HK is auto-generated and transferred to the conserved Asp residue on its cognate RR protein to elicit adaptive responses. A number of TCSs are integrated and required for persistence in response to a wide range of stressors and environments and for providing virulence in host cells [2,3,4]. TCSs are ubiquitous in bacteria but absent in mammals, so bacterial TCSs are potent targets for drug design, especially those that control virulence such as the PmrA/PmrB TCS [5]
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