An in-membrane NMR spectroscopic approach probing native ligand-GPCR interaction

Wang, Xudong; Bushra, Nabila; Muschol, Martin; Madsen, Jesper J.; Ye, Libin

doi:10.1016/j.ijbiomac.2022.03.099

Cited by 7 publications

(3 citation statements)

References 39 publications

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“…Thus, accurate measurements of dynamic properties of GPCRs are essential in complementing structural information. Experimental techniques such as nuclear magnetic resonance (NMR) spectroscopy can be used to determine ensembles of conformational states, 27 , 28 , 36 , 37 , 38 , 39 , 40 , 41 , 42 and hydrogen‐deuterium exchange‐mass spectroscopy ( HDX‐MS) has been used to determine changes in the relative solvent accessibility of amide protons to reveal specific alterations in the energy landscapes of the receptors conformational states. 29 , 43 , 44 , 45 …”

Section: Introductionmentioning

confidence: 99%

“…Thus, accurate measurements of dynamic properties of GPCRs are essential in complementing structural information. Experimental techniques such as nuclear magnetic resonance (NMR) spectroscopy can be used to determine ensembles of conformational states, 27,28,[36][37][38][39][40][41][42] and hydrogendeuterium exchange-mass spectroscopy (HDX-MS) has been used to determine changes in the relative solvent accessibility of amide protons to reveal specific alterations in the energy landscapes of the receptors conformational states. 29,[43][44][45] An informative way to model the flexibility present in a crystallized protein is to represent the structure as a set of overlapping, noninteracting conformers each accounting for a fraction of the total electron density.…”

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confidence: 99%

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Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures

Madsen

Frimurer

et al. 2022

Protein Science

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G protein‐coupled receptors (GPCRs) are important drug targets characterized by a canonical seven transmembrane (TM) helix architecture. Recent advances in X‐ray crystallography and cryo‐EM have resulted in a wealth of GPCR structures that have been used in drug design and formed the basis for mechanistic activation hypotheses. Here, ensemble refinement (ER) of crystallographic structures is applied to explore the impact of binding of agonists and antagonist/inverse agonists to selected structures of cannabinoid receptor 1 (CB1R), β2 adrenergic receptor (β2AR), and A2A adenosine receptor (A2AAR). To assess the conformational flexibility and its role in GPCR activation, hydrogen bond (H‐bond) networks are analyzed by calculating and comparing H‐bond propensities. Mapping pairwise propensity differences between agonist‐ and inverse agonist/antagonist‐bound structures for CB1R and β2AR shows that agonist binding destabilizes H‐bonds in the intracellular parts of TM 5–7, forming the G protein binding cavity, while H‐bonds of the extracellular segment of TMs surrounding the orthosteric site are conversely stabilized. Certain class A GPCRs, for example, A2AAR, bind an allosteric sodium ion that negatively modulates agonist binding. The impact of sodium‐excluding mutants (D522.50N, S913.39A) of A2AAR on agonist binding is examined by applying ER analysis to structures of wildtype and the two mutants in complex with a full agonist. While S913.39A exhibits normal activity, D522.50N quenches the downstream signaling. The mainchain H‐bond pattern of the latter is stabilized in the intracellular part of TM 7 containing the NPxxY motif, indicating that an induced rigidity of the mutation prevents conformational selection of G proteins resulting in receptor inactivation.

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Section: Introductionmentioning

confidence: 99%

“…Thus, accurate measurements of dynamic properties of GPCRs are essential in complementing structural information. Experimental techniques such as nuclear magnetic resonance (NMR) spectroscopy can be used to determine ensembles of conformational states, 27,28,[36][37][38][39][40][41][42] and hydrogendeuterium exchange-mass spectroscopy (HDX-MS) has been used to determine changes in the relative solvent accessibility of amide protons to reveal specific alterations in the energy landscapes of the receptors conformational states. 29,[43][44][45] An informative way to model the flexibility present in a crystallized protein is to represent the structure as a set of overlapping, noninteracting conformers each accounting for a fraction of the total electron density.…”

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confidence: 99%

Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures

Madsen

Frimurer

et al. 2022

Protein Science

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“…The structural characterization of GPCRs by X-ray crystallography and cryogenic electron microscopy (cryo-EM) has provided unprecedented insights into the processes of receptor activation [1][2][3][4][5][6] . However, our comprehension of GPCR signaling remains limited because these techniques typically capture low-energy states, whereas the full articulation of diversity and complexity in GPCR signaling requires descriptions based on dynamic ensembles in which conformational changes are continuous.…”

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confidence: 99%

Intermediate-state-trapped mutants pinpoint G protein-coupled receptor conformational allostery

et al. 2023

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Understanding the roles of intermediate states in signaling is pivotal to unraveling the activation processes of G protein-coupled receptors (GPCRs). However, the field is still struggling to define these conformational states with sufficient resolution to study their individual functions. Here, we demonstrate the feasibility of enriching the populations of discrete states via conformation-biased mutants. These mutants adopt distinct distributions among five states that lie along the activation pathway of adenosine A2A receptor (A2AR), a class A GPCR. Our study reveals a structurally conserved cation-π lock between transmembrane helix VI (TM6) and Helix8 that regulates cytoplasmic cavity opening as a “gatekeeper” for G protein penetration. A GPCR activation process based on the well-discerned conformational states is thus proposed, allosterically micro-modulated by the cation-π lock and a previously well-defined ionic interaction between TM3 and TM6. Intermediate-state-trapped mutants will also provide useful information in relation to receptor-G protein signal transduction.

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Solution NMR investigations of integral membrane proteins: Challenges and innovations

Necelis,

McDermott,

Belcher Dufrisne

et al. 2023

Current Opinion in Structural Biology

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An in-membrane NMR spectroscopic approach probing native ligand-GPCR interaction

Cited by 7 publications

References 39 publications

Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures

Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures

Intermediate-state-trapped mutants pinpoint G protein-coupled receptor conformational allostery

Solution NMR investigations of integral membrane proteins: Challenges and innovations

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