Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies.
CXCR4
NanoBiT
NanoLuciferase
allosterism
chemokine receptor
extracellular loop 2
nanobodies
Journal
Cell chemical biology
ISSN: 2451-9448
Titre abrégé: Cell Chem Biol
Pays: United States
ID NLM: 101676030
Informations de publication
Date de publication:
15 10 2020
15 10 2020
Historique:
received:
28
03
2020
revised:
01
06
2020
accepted:
12
06
2020
pubmed:
2
7
2020
medline:
7
7
2021
entrez:
2
7
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32610042
pii: S2451-9456(20)30229-4
doi: 10.1016/j.chembiol.2020.06.006
pmc: PMC7573392
pii:
doi:
Substances chimiques
CXCR4 protein, human
0
Receptors, CXCR4
0
Single-Domain Antibodies
0
Luciferases
EC 1.13.12.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1250-1261.e5Subventions
Organisme : Medical Research Council
ID : MR/N020081/1
Pays : United Kingdom
Informations de copyright
Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Interests R.H. is CSO of QVQ Holding B.V.
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