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Conformation space of a heterodimeric ABC exporter under turnover conditions

Author

Listed:
  • Susanne Hofmann

    (Goethe University Frankfurt)

  • Dovile Januliene

    (Max Planck Institute of Biophysics)

  • Ahmad R. Mehdipour

    (Max Planck Institute of Biophysics)

  • Christoph Thomas

    (Goethe University Frankfurt)

  • Erich Stefan

    (Goethe University Frankfurt)

  • Stefan Brüchert

    (Goethe University Frankfurt)

  • Benedikt T. Kuhn

    (Goethe University Frankfurt)

  • Eric R. Geertsma

    (Goethe University Frankfurt)

  • Gerhard Hummer

    (Max Planck Institute of Biophysics
    Goethe University Frankfurt)

  • Robert Tampé

    (Goethe University Frankfurt)

  • Arne Moeller

    (Max Planck Institute of Biophysics)

Abstract

Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action1–3. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol4–6 and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance7,8. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations—one of them with a bound peptide substrate—and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation—an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.

Suggested Citation

  • Susanne Hofmann & Dovile Januliene & Ahmad R. Mehdipour & Christoph Thomas & Erich Stefan & Stefan Brüchert & Benedikt T. Kuhn & Eric R. Geertsma & Gerhard Hummer & Robert Tampé & Arne Moeller, 2019. "Conformation space of a heterodimeric ABC exporter under turnover conditions," Nature, Nature, vol. 571(7766), pages 580-583, July.
  • Handle: RePEc:nat:nature:v:571:y:2019:i:7766:d:10.1038_s41586-019-1391-0
    DOI: 10.1038/s41586-019-1391-0
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    Cited by:

    1. Jun Gyou Park & Songwon Kim & Eunhong Jang & Seung Hun Choi & Hyunsu Han & Seulgi Ju & Ji Won Kim & Da Sol Min & Mi Sun Jin, 2022. "The lysosomal transporter TAPL has a dual role as peptide translocator and phosphatidylserine floppase," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
    2. Martin F. Peter & Christian Gebhardt & Rebecca Mächtel & Gabriel G. Moya Muñoz & Janin Glaenzer & Alessandra Narducci & Gavin H. Thomas & Thorben Cordes & Gregor Hagelueken, 2022. "Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET," Nature Communications, Nature, vol. 13(1), pages 1-19, December.
    3. Chancievan Thangaratnarajah & Mark Nijland & Luís Borges-Araújo & Aike Jeucken & Jan Rheinberger & Siewert J. Marrink & Paulo C. T. Souza & Cristina Paulino & Dirk J. Slotboom, 2023. "Expulsion mechanism of the substrate-translocating subunit in ECF transporters," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    4. Jiao Li & Wan Zheng & Ming Gu & Long Han & Yanmei Luo & Koukou Yu & Mengxin Sun & Yuliang Zong & Xiuxiu Ma & Bing Liu & Ethan P. Lowder & Deanna L. Mendez & Robert G. Kranz & Kai Zhang & Jiapeng Zhu, 2022. "Structures of the CcmABCD heme release complex at multiple states," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    5. Qingyu Tang & Matt Sinclair & Hale S. Hasdemir & Richard A. Stein & Erkan Karakas & Emad Tajkhorshid & Hassane S. Mchaourab, 2023. "Asymmetric conformations and lipid interactions shape the ATP-coupled cycle of a heterodimeric ABC transporter," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    6. Søren K. Amstrup & Sui Ching Ong & Nicholas Sofos & Jesper L. Karlsen & Ragnhild B. Skjerning & Thomas Boesen & Jan J. Enghild & Bjarne Hove-Jensen & Ditlev E. Brodersen, 2023. "Structural remodelling of the carbon–phosphorus lyase machinery by a dual ABC ATPase," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
    7. Mathieu Botte & Dongchun Ni & Stephan Schenck & Iwan Zimmermann & Mohamed Chami & Nicolas Bocquet & Pascal Egloff & Denis Bucher & Matilde Trabuco & Robert K. Y. Cheng & Janine D. Brunner & Markus A. , 2022. "Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation," Nature Communications, Nature, vol. 13(1), pages 1-10, December.

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