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

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.

Figure 1

Ligand dependent effects on the sodium bound states of A2aR. (A) Schematic illustrating the sodium binding pocket of A2aR in an inactive conformation (PDB 4EIY) and the collapse of the pocket upon adopting an active conformation (PDB 3QAK). 13+ charge state of A2aR electrosprayed from (B) 200 mM NH₄OAc and (C) 50 mM NaCl, 5 mM Tris (both pH 7.5 with 2× CMC LMNG) in the absence or presence of 10 μM ligand. Spectra are representative of n ≥ 4 nanoemitters and 2 protein preparations.

Figure 2

Preservation of lipophilic drug binding to the glucagon receptor in NaCl-containing electrospray buffers. (A) Structure of the full-length glucagon receptor (PDB 5XEZ) showing the allosteric binding pocket binding NNC0640, a variant of to NNC0666, situated between transmembrane loops VI and VII with interacting residues highlighted in blue. Native mass spectra of GCGR released from G1/CHS micelles in (B) 250 mM NH₄OAc, pH 7.5 electrospray buffer or (C) 50 mM NaCl, 5 mM Tris, pH 7.5 electrospray buffers with insets expanding the 13+ charge state. Differences in binding of the NNC066 are more prevalent in (C), highlighted orange and red for one and two NAMs, respectively. Spectra are representative of n ≥ 4 nanoemitters and n = 2 protein preparations.