Journal of Biological Chemistry
Volume 295, Issue 51, 18 December 2020, Pages 17865-17876
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Protein Structure and Folding
Antibiotic binding releases autoinhibition of the TipA multidrug-resistance transcriptional regulator

https://doi.org/10.1074/jbc.RA120.016295Get rights and content
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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.

multidrug resistance (MDR)
transcriptional regulator
TipA
autoinhibition
drug resistance
antibiotic resistance
transcription regulation
transcription promoter
DNA-binding protein
crystal structure
activation mechanism
structure biology

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This article contains supporting information.

Author contributions—X. J. and X. L. conceptualization; X. J. and L. Z. data curation; X. J. and L. Z. software; X. J., M. T., and X. L. supervision; X. J., L. Z., and X. L. validation; X. J. and L. Z. investigation; X. J. and X. L. writing-original draft; X. J., M. T., and X. L. project administration; X. J. and X. L. writing-review and editing; M. T. and X. L. funding acquisition; M. T. methodology; X. L. resources.

Funding and additional information—This work was supported by Chinese National Natural Science Foundation Grants 31971124, U1732114, 31770788, and 31770895 and National Key Research and Development Program of China Grant 2017YFA0503600.

Conflict of interest—The authors declare no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

    MD

    multidrug

    MDR

    multidrug resistance

    MtaN

    N terminus of Mta

    RNAP

    RNA polymerase

    MerR

    mercuric ion resistance

    HTH

    helix-turn-helix

    ITC

    isothermal titration calorimetry

    FPA

    fluorescence polarization assay

    SEC

    size-exclusion chromatography

    PDB

    Protein Data Bank

    FAM

    carboxyfluorescein

    SeMet

    selenomethionine.

These authors contributed equally to this work.