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Nanobody-enabled monitoring of kappa opioid receptor states
Che, T., English, J., Krumm, B. E., Kim, K., Pardon, E., Olsen, R. H. J., Wang, S., Zhang, S., Diberto, J. F., Sciaky, N., Carroll, F. I., Steyaert, J., Wacker, D., & Roth, B. L. (2020). Nanobody-enabled monitoring of kappa opioid receptor states. Nature Communications, 11(1), Article 1145. https://doi.org/10.1038/s41467-020-14889-7
Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.
Recent studies revealed that G protein-coupled receptors rapidly interconvert between multiple states. Here, authors use the kappa opioid receptor (KOR) and show how two state-dependent nanobodies provide real-time reporting of ligand stabilized states with KOR and other GPCRs.
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