The Effects of Sodium Ions on Ligand Binding and Conformational States of G Protein-Coupled Receptors-Insights from Mass Spectrometry.

Mark T Agasid,Leonhard H Urner,Jun Yan,Carol V Robinson,Lars Sørensen

doi:10.1021/jacs.0c11837

Abstract

The use of mass spectrometry to investigate proteins is now well established and provides invaluable information for both soluble and membrane protein assemblies. Maintaining transient noncovalent interactions under physiological conditions, however, remains challenging. Here, using nanoscale electrospray ionization emitters, we establish conditions that enable mass spectrometry of two G protein-coupled receptors (GPCR) from buffers containing high concentrations of sodium ions. For the Class A GPCR, the adenosine 2A receptor, we observe ligand-induced changes to sodium binding of the receptor at the level of individual sodium ions. We find that antagonists promote sodium binding while agonists attenuate sodium binding. These findings are in line with high-resolution X-ray crystallography wherein only inactive conformations retain sodium ions in allosteric binding pockets. For the glucagon receptor (a Class B GPCR) we observed enhanced ligand binding in electrospray buffers containing high concentrations of sodium, as opposed to ammonium acetate buffers. A combination of native and -omics mass spectrometry revealed the presence of a lipophilic negative allosteric modulator. These experiments highlight the advantages of implementing native mass spectrometry, from electrospray buffers containing high concentrations of physiologically relevant salts, to inform on allosteric ions or ligands with the potential to define their roles on GPCR function.

Highlights

The use of mass spectrometry to investigate proteins is well established and provides invaluable information for both soluble and membrane protein assemblies
These experiments highlight the advantages of implementing native mass spectrometry, from electrospray buffers containing high concentrations of physiologically relevant salts, to inform on allosteric ions or ligands with the potential to define their roles on G protein-coupled receptors (GPCR) function
Native mass spectrometry has become a powerful biophysical tool to characterize membrane proteins, including relatively few examples of GPCRs.[12,13] nMS exploits the gentle ionization conditions enabled by nanoelectrospray ionization,[14] which can preserve noncovalent interactions, informing on protein−ligand and protein−protein interactions.[15]

Summary

Introduction

The use of mass spectrometry to investigate proteins is well established and provides invaluable information for both soluble and membrane protein assemblies. For the glucagon receptor (a Class B GPCR) we observed enhanced ligand binding in electrospray buffers containing high concentrations of sodium, as opposed to ammonium acetate buffers.

Results

Conclusion