Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Allosteric Site
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Binding Sites
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Biosensing Techniques
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Crystallography, X-Ray
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Cyclic AMP / metabolism
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Dynorphins / chemistry
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Dynorphins / pharmacology
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HEK293 Cells
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Humans
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Luminescent Measurements / methods
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Piperazines / chemistry
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Piperazines / pharmacology
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Piperidines / chemistry
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Piperidines / pharmacology
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Protein Conformation
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Pyrrolidines / chemistry
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Pyrrolidines / pharmacology
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Receptors, G-Protein-Coupled / chemistry
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Receptors, G-Protein-Coupled / metabolism
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Receptors, Opioid, kappa / agonists
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Receptors, Opioid, kappa / chemistry*
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Receptors, Opioid, kappa / genetics
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Receptors, Opioid, kappa / metabolism*
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Single-Domain Antibodies / chemistry*
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Single-Domain Antibodies / metabolism
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Tetrahydroisoquinolines / chemistry
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Tetrahydroisoquinolines / pharmacology
Substances
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7-hydroxy-N-(1-((4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl)methyl)-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide
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Piperazines
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Piperidines
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Pyrrolidines
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Receptors, G-Protein-Coupled
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Receptors, Opioid, kappa
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Recombinant Proteins
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Single-Domain Antibodies
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Tetrahydroisoquinolines
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GR 89696
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Dynorphins
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Cyclic AMP