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Ligand induced conformational changes in the B1-Adrenergic Receptor Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry

ABSTRACT: Project employ Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), to characterise the structural dynamics of turkey β1-adrenergic receptor (tβ1AR) in complex with nine ligands, including agonists, partial agonists and antagonists.We show that dynamic signatures across the GPCR structure can be grouped by compound modality. Surprisingly, we discovered repeated destabilisation of the intracellular loop 1 (ICL1) upon full agonist binding and stabilisation upon antagonist binding, suggesting that increased dynamics in this region are an essential component for G protein recruitment. Multiple sequence alignments and molecular dynamics simulations indicate that L72 in ICL1 plays an important structural role. Differential HDX-MS experiment of tβ1AR and tβ1AR L72A construct in complex with miniGs, in response to various ligands, suggests involvement of ICL1 in stabilising the GDP bound state by influencing the stability of HG helix of miniGs.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Meleagris Gallopavo (common Turkey)

SUBMITTER: Joanna Toporowska

LAB HEAD: Argyris Politis

PROVIDER: PXD051297 | Pride | 2024-08-30

REPOSITORIES: Pride

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			Action	DRS
	090221_B1AR_dualp_2_IA_final_peptide.csv	Csv
	B1AR_Raw_Data_Repository_04042024.7z	Other

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Project description:Post expression from the host cell, a bio-therapeutic will be subjected to several downstream processing steps prior to final formulation. Analysis of the product under various buffer conditions at each stage is desirable to show that the integrity of the sample is maintained and to monitor any potential product loss. In this study we examine how varying buffer conditions during downstream processing impact the structural integrity of a biopharmaceutical product, with a specific focus on the model IgG1 antibody, mAb4, provided by FUJIFILM Diosynth Biotechnologies. Flexibility, stability, aggregation propensity, and bulk properties were evaluated across the buffers of four sample points, including perfusion media, purification stages, and formulation buffer. Comparisons with Herceptin, an extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible buffer. Although mAb4 and Herceptin present with similar charge state distributions (CSD) in native mass spectrometry (MS) experiments, they exhibit distinct unfolding patterns during the activated ion mobility-mass spectrometry (aIM-MS) and differential scanning fluorimetry (DSF). The greater structural stability and aggregation onset temperature (Tagg) observed in Herceptin, are primarily attributed to the heavier glycosylation and kappa-class light chains present in Herceptin compared to the lambda-class light chains in mAb4. Hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that while mAb4 experiences substantial structural changes during purification, characterized by high flexibility, a low melting temperature (Tm), and prevalent repulsive protein-protein interactions, it eventually transitions to a highly compact and stable structure in buffers with higher salt concentrations and during formulation. Notably, in the formulated environment, the third constant domain (CH3) of the heavy chain retains flexibility and is identified as a region of interest for aggregation formation. Here we showcase the integration of MS and orthogonal techniques to attain comprehensive information which can be utilized early in the development stage to aid in decision-making regarding targeted mutations or to guide the design space of bioprocesses and formulation choices.

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Ligand induced conformational changes in the B1-Adrenergic Receptor Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry

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