1H, 13C and 15N backbone chemical shift assignments of camelid single-domain antibodies against active state µ-opioid receptor

Sounier, Rémy; Yang, Yinshan; Hagelberger, Joanna; Granier, Sébastien; Déméné, Hélène

doi:10.1007/s12104-017-9733-z

Cited by 5 publications

(2 citation statements)

References 15 publications

(17 reference statements)

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“…The Nb33 was expressed and purified as described in our previous work (Sounier et al, 2017). Briefly, The DNA sequence of Nb33 was subcloned into a pMalp2x vector containing an N-terminal, 3C protease-cleavable maltose binding protein (MBP) tag and a C-terminal 8 × His tag.…”

Section: Nanobody Nb33 Expression and Purificationmentioning

confidence: 99%

Molecular insights into the biased signaling mechanism of the μ-opioid receptor

et al. 2021

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GPCR functional selectivity whereby a ligand discriminates specific signaling pathways has opened new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the β-arrestin pathways through the µ-opioid receptor (µOR). We combined functional assays in living cells, solution NMR spectroscopy and enhanced-sampling molecular dynamic simulations to identify the specific µOR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs. Most strikingly, the biased agonists triggered µOR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair β-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.

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Section: Nanobody Nb33 Expression and Purificationmentioning

confidence: 99%

Molecular insights into the biased signaling mechanism of the μ-opioid receptor

et al. 2021

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“…Expression of the isotopically labeled Gα subunit of the trimeric G protein complex has been carried out in E. coli , permitting deuteration and uniform or amino acid-specific labeling of G proteins 256 . Isotopically labeled G protein mimicking camelid single-chain antibodies, so-called “nanobodies,” for the μ-opioid receptor (MOR) have also been expressed and characterized by solution NMR 257 .…”

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confidence: 99%

GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures

Shimada

Ueda

Kofuku

et al. 2018

Nat Rev Drug Discov

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The 826 G protein-coupled receptors (GPCRs) in the human proteome regulate key physiological processes, and thus have long been attractive as drug targets. With crystal structure determinations of more than 50 different human GPCRs during the last decade, an initial platform for structure-based rational design has been established for drugs that target GPCRs, which is currently being augmented with cryo-EM structures of higher-order GPCR complexes. Nuclear magnetic resonance (NMR) spectroscopy in solution is one of the key approaches for expanding this platform with dynamic features, which can be accessed at physiological temperature and with minimal modification of the wild-type GPCR covalent structures. Here, we review strategies for the use of advanced biochemistry and NMR techniques with GPCRs, survey projects where crystal or cryo-EM structures have been complemented with NMR investigations, and discuss the impact of this integrative approach on GPCR biology and drug discovery. More than 30% of all drugs approved by the US Food and Drug Administration target G protein-coupled receptors (GPCRs)1–3, and these drugs are utilized in a wide range of therapeutic areas, including inflammation and diseases of the central nervous system as well as the cardiovascular, respiratory and gastrointestinal systems2,4. Currently more than 300 agents are in clinical trials, of which around 60 target novel GPCRs for which no drug has as yet been approved2. The novel GPCR targets also include orphan GPCRs, for which endogenous ligands have not yet been discovered2. Overall, the drugs approved so far target only 27% of the human non-olfactory GPCRs2, indicating that much excitement still lies ahead. Identifying new GPCR drugs will need additional detailed knowledge of GPCR biology, especially knowledge from structural biology, given the complex structure–function relationships involved in GPCR signaling. Along this line, recent reviews on GPCRs have covered studies with antibodies5 and nanobodies6, allosteric modulation7–10 biased signaling11–15, methods in GPCR structural biology16–19, GPCR crystal structures20–27 and drug development2,4,28,29, with some reviews addressing specific GPCR families30,31. Complementing the substantial number of GPCR crystal structures that have become available in the past decade, as well as the recent demonstrations of the potential for cryo-EM to provide information on higher-order GPCR complexes32–36, dynamic studies of GPCRs are important for providing new insights into GPCR biology that can assist drug discovery. In this respect, nuclear magnetic resonance (NMR) spectroscopy in solution is a key tool for analysing function-related conformational equilibria in GPCRs as they relate to allosteric coupling, variable efficacies and biased signaling of GPCR ligands, which are of particular interest for their potential as drugs. Furthermore, NMR spectroscopy is also a useful tool for fragment-based lead discovery with GPCR targets. In this article, we first overview the structural biology of GPCRs ba...

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