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Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

Author

Listed:
  • Somnath Mukherjee

    (The University of Chicago)

  • Satchal K. Erramilli

    (The University of Chicago)

  • Mark Ammirati

    (Medicine Design, Worldwide Research and Development, Pfizer Inc.)

  • Frances J. D. Alvarez

    (Medicine Design, Worldwide Research and Development, Pfizer Inc.)

  • Kimberly F. Fennell

    (Medicine Design, Worldwide Research and Development, Pfizer Inc.)

  • Michael D. Purdy

    (University of Virginia School of Medicine)

  • Blazej M. Skrobek

    (The University of Chicago
    Ludwig Maximilian University of Munich)

  • Katarzyna Radziwon

    (The University of Chicago
    University of Wisconsin Madison)

  • John Coukos

    (The University of Chicago)

  • Yanyong Kang

    (Center for Cancer and Cell Biology, Structural Biology Program, Van Andel Research Institute
    Takeda San Diego Inc.)

  • Przemysław Dutka

    (The University of Chicago
    California Institute of Technology)

  • Xiang Gao

    (Center for Cancer and Cell Biology, Structural Biology Program, Van Andel Research Institute)

  • Xiayang Qiu

    (Medicine Design, Worldwide Research and Development, Pfizer Inc.)

  • Mark Yeager

    (University of Virginia School of Medicine)

  • H. Eric Xu

    (Center for Cancer and Cell Biology, Structural Biology Program, Van Andel Research Institute
    Center for Structure and Function of Drug Targets, Shanghai Institute of Materia Medica, Chinese Academy of Sciences)

  • Seungil Han

    (Medicine Design, Worldwide Research and Development, Pfizer Inc.)

  • Anthony A. Kossiakoff

    (The University of Chicago
    University of Chicago)

Abstract

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

Suggested Citation

  • Somnath Mukherjee & Satchal K. Erramilli & Mark Ammirati & Frances J. D. Alvarez & Kimberly F. Fennell & Michael D. Purdy & Blazej M. Skrobek & Katarzyna Radziwon & John Coukos & Yanyong Kang & Przemy, 2020. "Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins," Nature Communications, Nature, vol. 11(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-15363-0
    DOI: 10.1038/s41467-020-15363-0
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    Cited by:

    1. Kaihua Zhang & Hao Wu & Nicholas Hoppe & Aashish Manglik & Yifan Cheng, 2022. "Fusion protein strategies for cryo-EM study of G protein-coupled receptors," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    2. Aurélien Fouillen & Julien Bous & Pierre Couvineau & Hélène Orcel & Charline Mary & Lucie Lafleur & Timothé Pierre & Christiane Mendre & Nicolas Gilles & Gunnar Schulte & Sébastien Granier & Bernard M, 2025. "Inactive structures of the vasopressin V2 receptor reveal distinct binding modes for Tolvaptan and Mambaquaretin toxin," Nature Communications, Nature, vol. 16(1), pages 1-15, December.
    3. Nitesh Kumar Khandelwal & Meghna Gupta & James E. Gomez & Sulyman Barkho & Ziqiang Guan & Ashley Y. Eng & Tomohiko Kawate & Sree Ganesh Balasubramani & Andrej Sali & Deborah T. Hung & Robert M. Stroud, 2025. "Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporter efflux protein A," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    4. David Jonathan Wasilko & Brian S. Gerstenberger & Kathleen A. Farley & Wei Li & Jennifer Alley & Mark E. Schnute & Ray J. Unwalla & Jorge Victorino & Kimberly K. Crouse & Ru Ding & Parag V. Sahasrabud, 2024. "Structural basis for CCR6 modulation by allosteric antagonists," Nature Communications, Nature, vol. 15(1), pages 1-13, December.

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