Native Mass Spectrometry Reveals Ligand-Specific Conformational Heterogeneity of the β1-Adrenergic Receptor Using Nanobody Probes.
- 1Institute of Biological Chemistry, Academia Sinica, Taipei115201, Taiwan.
- 2Institute of Biochemical Sciences, National Taiwan University, Taipei106319, Taiwan.
- 3Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei115201, Taiwan.
Abstract
G protein-coupled receptors (GPCRs) are central physiological signal transducers and represent a primary class of therapeutic targets. While emerging evidence highlights a critical link between ligand-induced receptor conformations and pharmacological outcomes, the structural diversity dictating GPCR-G protein interface interactions remains incompletely understood. High-resolution structural studies reveal discrete, ligand-stabilized states, but these static snapshots do not fully capture the dynamic processes underlying GPCR activation and ligand efficacy. To address this, we established a native mass spectrometry (nMS) platform to probe ligand-induced conformational heterogeneity at the GPCR intracellular interface. Using the β1-adrenergic receptor (β1AR) as a model, we demonstrated a strong correlation between ligand efficacy and receptor complex formation with diverse active-state-specific nanobodies. Notably, leveraging the quantitative capacity of nMS, we uncovered distinct propensities of two highly similar nanobodies, Nb80 and Nb6B9, and showed that Nb6B9 engages weak-agonist-stabilized receptor states more effectively than Nb80. These interactions indicate that Nb6B9 recognizes ligand-specific conformations distinct from active states induced by full agonists. To gain structural insights, we utilized Nb6B9 chimeras with exchanged complementarity-determining regions (CDRs) and identified CDR1 and CDR3 as the primary drivers for ligand-specific recognition, implicating the involvement of receptor transmembrane motifs TM5 and TM6 in this conformational diversity. Finally, we demonstrated that nanobody-based nMS can distinguish receptor responses induced by the two salbutamol enantiomers, implying stereochemistry-dependent intracellular interface heterogeneity. Together, our study highlights the utility of nanobody-based nMS for evaluating the structural heterogeneity and dynamic landscapes of GPCRs.
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BibTeX
@article{wang2026native,
title = {{Native Mass Spectrometry Reveals Ligand-Specific Conformational Heterogeneity of the β1-Adrenergic Receptor Using Nanobody Probes.}},
author = {Yi-Quan Wang and Guan-Ting Lian and Yi-An Chen and Rong Chen and Peng-Yi Wu and Hsiang-Hui Peng and Hsin-Yung Yen},
journal = {Analytical chemistry},
year = {2026},
month = oct,
doi = {10.1021/acs.analchem.6c04571},
url = {https://europepmc.org/article/MED/42916126},
note = {CC BY},
}
RIS
TY - JOUR
TI - Native Mass Spectrometry Reveals Ligand-Specific Conformational Heterogeneity of the β1-Adrenergic Receptor Using Nanobody Probes.
AU - Yi-Quan Wang
AU - Guan-Ting Lian
AU - Yi-An Chen
AU - Rong Chen
AU - Peng-Yi Wu
AU - Hsiang-Hui Peng
AU - Hsin-Yung Yen
PY - 2026
DA - 2026/10/01
JO - Analytical chemistry
DO - 10.1021/acs.analchem.6c04571
UR - https://europepmc.org/article/MED/42916126
AB - G protein-coupled receptors (GPCRs) are central physiological signal transducers and represent a primary class of therapeutic targets. While emerging evidence highlights a critical link between ligand-induced receptor conformations and pharmacological outcomes, the structural diversity dictating GPCR-G protein interface interactions remains incompletely understood. High-resolution structural studies reveal discrete, ligand-stabilized states, but these static snapshots do not fully capture the dynamic processes underlying GPCR activation and ligand efficacy. To address this, we established a native mass spectrometry (nMS) platform to probe ligand-induced conformational heterogeneity at the GPCR intracellular interface. Using the β1-adrenergic receptor (β1AR) as a model, we demonstrated a strong correlation between ligand efficacy and receptor complex formation with diverse active-state-specific nanobodies. Notably, leveraging the quantitative capacity of nMS, we uncovered distinct propensities of two highly similar nanobodies, Nb80 and Nb6B9, and showed that Nb6B9 engages weak-agonist-stabilized receptor states more effectively than Nb80. These interactions indicate that Nb6B9 recognizes ligand-specific conformations distinct from active states induced by full agonists. To gain structural insights, we utilized Nb6B9 chimeras with exchanged complementarity-determining regions (CDRs) and identified CDR1 and CDR3 as the primary drivers for ligand-specific recognition, implicating the involvement of receptor transmembrane motifs TM5 and TM6 in this conformational diversity. Finally, we demonstrated that nanobody-based nMS can distinguish receptor responses induced by the two salbutamol enantiomers, implying stereochemistry-dependent intracellular interface heterogeneity. Together, our study highlights the utility of nanobody-based nMS for evaluating the structural heterogeneity and dynamic landscapes of GPCRs.
SN - 0003-2700
SN - 1520-6882
ER -