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.

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: 2451-9456
• 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