2026-10-13T13:02:39Z https://api.figshare.com/v2/oai

oai:figshare.com:article/34003060 2026-09-26T14:11:53Z category_1 category_4 category_915 category_14 category_19 category_21 category_873 category_734 portal_63 item_type_3 month_year_09_2026

Native Mass Spectrometry Reveals Ligand-Specific Conformational Heterogeneity of the β₁‑Adrenergic Receptor Using Nanobody Probes Yi-Quan Wang (19792280) Guan-Ting Lian (23787829) Yi-An Chen (83967) Rong Chen (29176) Peng-Yi Wu (25114525) Hsiang-Hui Peng (25114528) Hsin-Yung Yen (2256853) Biophysics Biochemistry Physical Sciences not elsewhere classified Molecular Biology Pharmacology Biotechnology Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified uncovered distinct propensities two salbutamol enantiomers native mass spectrometry gpcr intracellular interface receptor complex formation induced receptor conformations utilized nb6b9 chimeras specific conformations distinct nb6b9 engages weak gain structural insights emerging evidence highlights induced conformational heterogeneity active states induced stabilized receptor states specific conformational heterogeneity 1 </ sub nb6b9 recognizes ligand structural heterogeneity stabilized states adrenergic receptor conformational diversity study highlights specific recognition specific nanobodies diverse active therapeutic targets strong correlation static snapshots quantitative capacity primary drivers primary class pharmacological outcomes interactions indicate implying stereochemistry identified cdr1 fully capture full agonists exchanged complementarity dynamic landscapes determining regions critical link coupled receptors 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. 2026-09-26T00:00:00Z Dataset Dataset 10.1021/acs.analchem.6c04571.s002 https://figshare.com/articles/dataset/Native_Mass_Spectrometry_Reveals_Ligand-Specific_Conformational_Heterogeneity_of_the_sub_1_sub_Adrenergic_Receptor_Using_Nanobody_Probes/34003060 CC BY-NC 4.0