Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Tomasz Uchański; Simonas Masiulis; Baptiste Fischer; Valentina Kalichuk; Uriel López-Sánchez; Eleftherios Zarkadas; Miriam Weckener; Andrija Sente; Philip Ward; Alexandre Wohlkönig; Thomas Zögg; Han Remaut; James H Naismith; Hugues Nury; Wim Vranken; A Radu Aricescu; Els Pardon; Jan Steyaert

doi:10.1038/s41592-020-01001-6

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Tomasz Uchański, Simonas Masiulis, Baptiste Fischer, Valentina Kalichuk, Uriel López-Sánchez, Eleftherios Zarkadas, Miriam Weckener, Andrija Sente, Philip Ward, Alexandre Wohlkönig, Thomas Zögg, Han Remaut, James H Naismith, Hugues Nury, Wim Vranken, A Radu Aricescu, Els Pardon, Jan Steyaert

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Abstract

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.

Original language	English
Pages (from-to)	60-68
Number of pages	9
Journal	Nature Methods
Volume	18
Issue number	1
DOIs	https://doi.org/10.1038/s41592-020-01001-6
Publication status	Published - Jan 2021

Bibliographical note

Funding Information:
We thank A.V. Shkumatov and R.K. Singh for support with SAXS experiments, and H. De Greve for providing the GFP+-expressing E. coli strain. We thank Instruct-ERIC, part of the European Strategy Forum on Research Infrastructures (ESFRI), Instruct-ULTRA (EU H2020 grant no. 731005) and the Research Foundation—Flanders (FWO) for support with nanobody discovery and for funding the PhD training of T.U. We thank E. Beke for the technical assistance during megabody recloning. We thank Diamond Light Source, Harwell, UK, for access to crystallographic beamlines I03 and I24, and SAXS beamline B21. Cryo-EM studies of GABAA receptor were supported by the UK Medical Research Council grants no. MR/L009609/1 and no. MC_UP_1201/15 to A.R.A. We thank S. Chen, G. Cannone, G. Sharov and A. Yeates for support at the MRC-LMB EM facility; and J. Grimmett, T. Darling and T. Pratt for help with IT and high-performance computing. Cryo-EM studies of 5-HT3A receptor were supported by the ERC Starting grant no. 637733 Pentabrain, and the Fondation pour la Recherche Médicale grant no. SPF201809007073 to U.L.-S. We thank G. Schoehn and the IBS electron microscopy facility, supported by the Rhône-Alpes Region, the FRM, the FEDER and the GIS-IBISA. Cryo-EM studies of WbaP transferase were performed at Oxford Particle Imaging Centre founded by a Wellcome Trust JIF award (grant no. 060208/Z/00/Z) and supported by equipment grants from WT (grant no. 093305/Z/10/Z). We thank B. Qureshi for support with electron microscopy.

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© 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.

Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.

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10.1038/s41592-020-01001-6

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Uchański, T., Masiulis, S., Fischer, B., Kalichuk, V., López-Sánchez, U., Zarkadas, E., Weckener, M., Sente, A., Ward, P., Wohlkönig, A., Zögg, T., Remaut, H., Naismith, J. H., Nury, H., Vranken, W., Aricescu, A. R., Pardon, E., & Steyaert, J. (2021). Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM. Nature Methods, 18(1), 60-68. https://doi.org/10.1038/s41592-020-01001-6

Uchański, Tomasz ; Masiulis, Simonas ; Fischer, Baptiste ; Kalichuk, Valentina ; López-Sánchez, Uriel ; Zarkadas, Eleftherios ; Weckener, Miriam ; Sente, Andrija ; Ward, Philip ; Wohlkönig, Alexandre ; Zögg, Thomas ; Remaut, Han ; Naismith, James H ; Nury, Hugues ; Vranken, Wim ; Aricescu, A Radu ; Pardon, Els ; Steyaert, Jan. / Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM. In: Nature Methods. 2021 ; Vol. 18, No. 1. pp. 60-68.

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abstract = "Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.",

author = "Tomasz Ucha{\'n}ski and Simonas Masiulis and Baptiste Fischer and Valentina Kalichuk and Uriel L{\'o}pez-S{\'a}nchez and Eleftherios Zarkadas and Miriam Weckener and Andrija Sente and Philip Ward and Alexandre Wohlk{\"o}nig and Thomas Z{\"o}gg and Han Remaut and Naismith, {James H} and Hugues Nury and Wim Vranken and Aricescu, {A Radu} and Els Pardon and Jan Steyaert",

note = "Funding Information: We thank A.V. Shkumatov and R.K. Singh for support with SAXS experiments, and H. De Greve for providing the GFP+-expressing E. coli strain. We thank Instruct-ERIC, part of the European Strategy Forum on Research Infrastructures (ESFRI), Instruct-ULTRA (EU H2020 grant no. 731005) and the Research Foundation—Flanders (FWO) for support with nanobody discovery and for funding the PhD training of T.U. We thank E. Beke for the technical assistance during megabody recloning. We thank Diamond Light Source, Harwell, UK, for access to crystallographic beamlines I03 and I24, and SAXS beamline B21. Cryo-EM studies of GABAA receptor were supported by the UK Medical Research Council grants no. MR/L009609/1 and no. MC_UP_1201/15 to A.R.A. We thank S. Chen, G. Cannone, G. Sharov and A. Yeates for support at the MRC-LMB EM facility; and J. Grimmett, T. Darling and T. Pratt for help with IT and high-performance computing. Cryo-EM studies of 5-HT3A receptor were supported by the ERC Starting grant no. 637733 Pentabrain, and the Fondation pour la Recherche M{\'e}dicale grant no. SPF201809007073 to U.L.-S. We thank G. Schoehn and the IBS electron microscopy facility, supported by the Rh{\^o}ne-Alpes Region, the FRM, the FEDER and the GIS-IBISA. Cryo-EM studies of WbaP transferase were performed at Oxford Particle Imaging Centre founded by a Wellcome Trust JIF award (grant no. 060208/Z/00/Z) and supported by equipment grants from WT (grant no. 093305/Z/10/Z). We thank B. Qureshi for support with electron microscopy. Publisher Copyright: {\textcopyright} 2021, The Author(s), under exclusive licence to Springer Nature America, Inc. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

year = "2021",

month = jan,

doi = "10.1038/s41592-020-01001-6",

language = "English",

volume = "18",

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Uchański, T, Masiulis, S, Fischer, B , Kalichuk, V, López-Sánchez, U, Zarkadas, E, Weckener, M, Sente, A, Ward, P, Wohlkönig, A , Zögg, T , Remaut, H, Naismith, JH, Nury, H, Vranken, W, Aricescu, AR, Pardon, E & Steyaert, J 2021, 'Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM', Nature Methods, vol. 18, no. 1, pp. 60-68. https://doi.org/10.1038/s41592-020-01001-6

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM. / Uchański, Tomasz; Masiulis, Simonas; Fischer, Baptiste ; Kalichuk, Valentina; López-Sánchez, Uriel; Zarkadas, Eleftherios; Weckener, Miriam; Sente, Andrija; Ward, Philip; Wohlkönig, Alexandre ; Zögg, Thomas ; Remaut, Han; Naismith, James H; Nury, Hugues; Vranken, Wim; Aricescu, A Radu; Pardon, Els ; Steyaert, Jan.

In: Nature Methods, Vol. 18, No. 1, 01.2021, p. 60-68.

Research output: Contribution to journal › Article › peer-review

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AU - Uchański, Tomasz

AU - Masiulis, Simonas

AU - Fischer, Baptiste

AU - Kalichuk, Valentina

AU - López-Sánchez, Uriel

AU - Zarkadas, Eleftherios

AU - Weckener, Miriam

AU - Sente, Andrija

AU - Ward, Philip

AU - Wohlkönig, Alexandre

AU - Zögg, Thomas

AU - Remaut, Han

AU - Naismith, James H

AU - Nury, Hugues

AU - Vranken, Wim

AU - Aricescu, A Radu

AU - Pardon, Els

AU - Steyaert, Jan

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PY - 2021/1

Y1 - 2021/1

N2 - Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.

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Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

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