Abstract
Investigations of bacterial resistance strategies can aid in the development of new antimicrobial drugs as a countermeasure to the increasing worldwide prevalence of bacterial antibiotic resistance. One such strategy involves the TipA class of transcription factors, which constitute minimal autoregulated multidrug resistance (MDR) systems against diverse antibiotics. However, we have insufficient information regarding how antibiotic binding induces transcriptional activation to design molecules that could interfere with this process. To learn more, we determined the crystal structure of SkgA from Caulobacter crescentus as a representative TipA protein. We identified an unexpected spatial orientation and location of the antibiotic-binding TipAS effector domain in the apo state. We observed that the α6–α7 region of the TipAS domain, which is canonically responsible for forming the lid of antibiotic-binding cleft to tightly enclose the bound antibiotic, is involved in the dimeric interface and stabilized via interaction with the DNA-binding domain in the apo state. Further structural and biochemical analyses demonstrated that the unliganded TipAS domain sterically hinders promoter DNA binding but undergoes a remarkable conformational shift upon antibiotic binding to release this autoinhibition via a switch of its α6–α7 region. Hence, the promoters for MDR genes including tipA and RNA polymerases become available for transcription, enabling efficient antibiotic resistance. These insights into the molecular mechanism of activation of TipA proteins advance our understanding of TipA proteins, as well as bacterial MDR systems, and may provide important clues to block bacterial resistance.
Highlights
Investigations of bacterial resistance strategies can aid in the development of new antimicrobial drugs as a countermeasure to the increasing worldwide prevalence of bacterial antibiotic resistance
The established structure consists of the N-terminal mercuric ion resistance (MerR)-type HTH motif, coiled-coil region, and the initial two a-helices of the C-terminal TipAS domain (Fig. 1B and Fig. S1B), whereas the rest of the TipAS domain was still invisible in the current model, probably because of structural flexibility or diffraction damage
Because the above structural and biochemical analyses indicated that the TipAS domain inhibited TipAN–DNA binding by directly hindering access of DNA to the HTH motif in the apo state, we further investigated how drug binding induces activation
Summary
Investigations of bacterial resistance strategies can aid in the development of new antimicrobial drugs as a countermeasure to the increasing worldwide prevalence of bacterial antibiotic resistance. Structural and biochemical analyses indicated that the interaction of TipA regulators with promoter DNA is hindered in the apo state and revealed an unexpected activation mechanism in which drug binding induces conformational turnover of
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