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Oxygen-Induced Conformational Changes in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor

  • Emilie Orillard
    Emilie Orillard
    Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, California 92350, United States
  • Selina Anaya
    Selina Anaya
    Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, California 92350, United States
    More by Selina Anaya
  • Mark S. Johnson
    Mark S. Johnson
    Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, California 92350, United States
  • , and 
  • Kylie J. Watts*
    Kylie J. Watts
    Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, California 92350, United States
    *Email: [email protected]
Cite this: Biochemistry 2021, 60, 34, 2610–2622
Publication Date (Web):August 12, 2021
https://doi.org/10.1021/acs.biochem.1c00452
Copyright © 2021 American Chemical Society

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    Abstract

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    The Aer2 receptor from Pseudomonas aeruginosa has an O2-binding PAS-heme domain that stabilizes O2 via a Trp residue in the distal heme pocket. Trp rotates ∼90° to bond with the ligand and initiate signaling. Although the isolated PAS domain is monomeric, both in solution and in a cyanide-bound crystal structure, an unliganded structure forms a dimer. An overlay of the two structures suggests possible signaling motions but also predicts implausible clashes at the dimer interface when the ligand is bound. Moreover, in a full-length Aer2 dimer, PAS is sandwiched between multiple N- and C-terminal HAMP domains, which would feasibly restrict PAS motions. To explore the PAS dimer interface and signal-induced motions in full-length Aer2, we introduced Cys substitutions and used thiol-reactive probes to examine in vivo accessibility and residue proximities under both aerobic and anaerobic conditions. In vivo, PAS dimers were retained in full-length Aer2 in the presence and absence of O2, and the dimer interface was consistent with the isolated PAS dimer structure. O2-mediated changes were also consistent with structural predictions in which the PAS N-terminal caps move apart and the C-terminal DxT region moves closer together. The DxT motif links PAS to the C-terminal HAMP domains and was critical for PAS-HAMP signaling. Removing the N-terminal HAMP domains altered the distal PAS dimer interface and prevented signaling, even after signal-on lesions were introduced into PAS. The N-terminal HAMP domains thus facilitate the O2-dependent shift of PAS to the signal-on conformation, clarifying their role upstream of the PAS-sensing domain.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.1c00452.

    • Aer2 PAS side chain solvent accessible surface areas (Table S1), structures and chemical reactions for PEG-mal and BMOE (Figure S1), steady-state cellular levels of Aer2 proteins (Figure S2), example behavioral assays (Figure S3), lack of PAS interactions in BACTH assays (Figure S4), and comparison of average extents of cross-linking for Aer21–679-Cys and Aer2173–679-Cys mutants exposed to BMOE (Figure S5) (PDF)

    Accession Codes

    P. aeruginosa Aer2 PAS monomer with cyanomet, Q9I6V6 (UniProtKB) and 3VOL (PDB); P. aeruginosa Aer2 PAS dimer, Q9I6V6 (UniProtKB) and 4HI4 (PDB); P. aeruginosa Aer2 HAMP domains 1–3, Q9I6V6 (UniProtKB) and 3LNR (PDB); Thermotoga maritima methyl-accepting chemotaxis protein (MCP)1143, Q9X0M7 (UniProtKB) and 2CH7 (PDB).

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    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

    Cited By

    This article is cited by 4 publications.

    1. Erwin C. Stuffle, Tise Suzuki, Emilie Orillard, Kylie J. Watts. The Aer2 chemoreceptor from Vibrio vulnificus is a tri‐PAS‐heme oxygen sensor. Molecular Microbiology 2023, 119 (1) , 59-73. https://doi.org/10.1111/mmi.15007
    2. Bibi Zhou, Christine M. Szymanski, Arden Baylink. Bacterial chemotaxis in human diseases. Trends in Microbiology 2022, 45 https://doi.org/10.1016/j.tim.2022.10.007
    3. Selina Anaya, Emilie Orillard, Suzanne E. Greer-Phillips, Kylie J. Watts, . New Roles for HAMP Domains: the Tri-HAMP Region of Pseudomonas aeruginosa Aer2 Controls Receptor Signaling and Cellular Localization. Journal of Bacteriology 2022, 204 (9) https://doi.org/10.1128/jb.00225-22
    4. Emilie Orillard, Kylie J. Watts, . Leptospira interrogans Aer2: an Unusual Membrane-Bound PAS-Heme Oxygen Sensor. Journal of Bacteriology 2022, 204 (4) https://doi.org/10.1128/jb.00567-21

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