Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies

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

doi:10.1016/j.chembiol.2020.06.006

Mark Soave, Raimond Heukers + Show 5 more

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https://doi.org/10.1016/j.chembiol.2020.06.006

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Abstract

SummaryCamelid 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.

Highlights

The C-X-C chemokine receptor 4 (CXCR4) is a family A G protein-coupled receptor (GPCR) that plays an important role in the immune response and in the progression of many diseases, including cancer and HIV infection (Kucia et al, 2004; Scholten et al, 2012)
Characterization of LgBiT-CXCR4 with NanoBiT As part of the necessary tool development, we initially used the NanoBiT technology to characterize the binding of VUN400 to CXCR4
HiBiT was added in the form of a purified HiBiT-HaloTag fusion protein, circa 34 kDa in size, which was too large to cross the plasma membrane

Summary

Introduction

The C-X-C chemokine receptor 4 (CXCR4) is a family A G protein-coupled receptor (GPCR) that plays an important role in the immune response and in the progression of many diseases, including cancer and HIV infection (Kucia et al, 2004; Scholten et al, 2012). Of the chemokine receptor family, CXCR4 is unusual in that it exclusively binds one chemokine ligand, CXCL12 (formerly termed stromal cell-derived factor 1a). CXCL12 binds to CXCR4 in a two-step process: first interacting with residues in the N terminus of the receptor (chemokine recognition site 1), before CXCL12 engages both the extracellular loops and binding pocket within the transmembrane helices (chemokine recognition site 2 [CR2]; Kofuku et al, 2009). The structure of CXCR4 has since been solved in the presence of the small-molecule antagonist IT1t, the cyclic peptide antagonist CVX15 (Wu et al, 2010), and the chemokine antagonist vMIP-II (Qin et al, 2015)

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