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

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, Przemysław Dutka, Xiang Gao, Xiayang Qiu, Mark Yeager, H. Eric Xu, Seungil Han and Anthony A. Kossiakoff ()
Additional contact information
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
Katarzyna Radziwon: The University of Chicago
John Coukos: The University of Chicago
Yanyong Kang: Center for Cancer and Cell Biology, Structural Biology Program, Van Andel Research Institute
Przemysław Dutka: The University of Chicago
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
Seungil Han: Medicine Design, Worldwide Research and Development, Pfizer Inc.
Anthony A. Kossiakoff: The University of Chicago

Nature Communications, 2020, vol. 11, issue 1, 1-14

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-15363-0

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