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Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies

Soave, Mark; Heukers, Raimond; Kellam, Barrie; Woolard, Jeanette; Smit, Martine J.; Briddon, Stephen J.; Hill, Stephen J.

Authors

Mark Soave

Raimond Heukers

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BARRIE KELLAM BARRIE.KELLAM@NOTTINGHAM.AC.UK
Professor of Medicinal Chemistry

JEANETTE ISAAC Jeanette.Woolard@nottingham.ac.uk
Professor of Cardiovascular Physiology and Pharmacology

Martine J. Smit

STEPHEN HILL steve.hill@nottingham.ac.uk
Professor of Molecular Pharmacology



Abstract

© 2020 The Authors Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15-kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognizes the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C terminus to the 11-amino-acid HiBiT tag (VUN400-HiBiT) which complements LgBiT protein, forming a full-length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect allosteric interactions with CXCR4. These data demonstrate that the high specificity offered by extracellular targeted nanobodies can be utilized to probe receptor pharmacology.

Citation

Soave, M., Heukers, R., Kellam, B., Woolard, J., Smit, M. J., Briddon, S. J., & Hill, S. J. (2020). Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies. Cell Chemical Biology, 27, 1-12. https://doi.org/10.1016/j.chembiol.2020.06.006

Journal Article Type Article
Acceptance Date Jun 12, 2020
Online Publication Date Jun 30, 2020
Publication Date 2020-06
Deposit Date Jun 24, 2020
Publicly Available Date Jul 1, 2020
Journal Cell Chemical Biology
Electronic ISSN 2451-9448
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 27
Pages 1-12
DOI https://doi.org/10.1016/j.chembiol.2020.06.006
Keywords Clinical Biochemistry; Molecular Medicine; Biochemistry; Molecular Biology; Pharmacology; Drug Discovery
Public URL https://nottingham-repository.worktribe.com/output/4705946
Publisher URL https://www.cell.com/cell-chemical-biology/fulltext/S2451-9456(20)30229-4

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