Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor

Abstract

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

Glycosylated Amphiphilic Calixarene‐Based Detergent for Functional Stabilization of Native Membrane Proteins.

“…A 2A R shows a smear signal. This is consistent with the presence of different non‐aggregated oligomers as recently reported . Native PAGE and SEC confirmed that AcrB, BmrA and A 2A R were not aggregated when DDM/CALX‐173‐GK were used for both solubilization and purification ( 5B ).…”

“…We then investigated the function of each solubilized and purified membrane protein. We compared CALX‐173‐GK condition to a reference as previously reported, (DDM for BmrA and AcrB, DDM/CHS for A 2A R). Very similar ligand binding activity was observed for AcrB in both solubilization and purification conditions.…”

Detergent‐Like Polymerizable Monomers: Synthesis, Physicochemical, and Biochemical Characterization

“…Solubilization of A 2A R and AcrB: Adenosine receptor (A 2A R) was expressed in insect cells as described . AcrB was expressed in E.coli as previously reported .…”

Multivalent and Multifunctional Calixarenes in Bionanotechnology

“…It was proposed and partially proved that these amphiphiles freeze the monomeric structure of the protein, preventing its aggregation and denaturation, through hydrophobic interactions with the protein membrane region and through a network of salt bridges with the basic residues at the cytosol-membrane interface. With the aim of overcoming what has been indicated as a bottleneck in the membrane protein structural biology, [75] in the latest years other studies appeared where the stabilisation, isolation and characterisation of membrane proteins such as Human Multidrug Resistance Protein 4 [76] and G protein-coupled receptors, [77] using similar calixarene detergents, were reported. A glycocalixarene having glucose units at the upper and an heptyl chain at the lower rim also showed to preserve and stabilise, even over long periods of time, the function of three native membrane proteins.…”