Real-time monitoring of binding events on a thermostabilized human A2A receptor embedded in a lipid bilayer by surface plasmon resonance

Abstract

Membrane proteins (MPs) are prevalent drug discovery targets involved in many cell processes. Despite their high potential as drug targets, the study of MPs has been hindered by limitations in expression, purification and stabilization in order to acquire thermodynamic and kinetic parameters of small molecules binding. These bottlenecks are grounded on the mandatory use of detergents to isolate and extract MPs from the cell plasma membrane and the coexistence of multiple conformations, which reflects biochemical versatility and intrinsic instability of MPs. In this work ,we set out to define a new strategy to enable surface plasmon resonance (SPR) measurements on a thermostabilized and truncated version of the human adenosine (A2A) G-protein-coupled receptor (GPCR) inserted in a lipid bilayer nanodisc in a label- and detergent-free manner by using a combination of affinity tags and GFP-based fluorescence techniques. We were able to detect and characterize small molecules binding kinetics on a GPCR fully embedded in a lipid environment. By providing a comparison between different binding assays in membranes, nanodiscs and detergent micelles, we show that nanodiscs can be used for small molecule binding studies by SPR to enhance the MP stability and to trigger a more native-like behaviour when compared to kinetics on A2A receptors isolated in detergent. This work provides thus a new methodology in drug discovery to characterize the binding kinetics of small molecule ligands for MPs targets in a lipid environment.

A2AR Binding Kinetics in the Ligand Depletion Regime

“…Specifically, the k on values we obtain for micelle-solubilized A 2A R are within ~10 fold of those for FITC-APEC 22 and 5–10 fold of those determined from ZM 241,385 and NECA using [ 3 H]-ZM 241,385 4 for wild-type A 2A R in membrane preparations. The k on values for ZM 241,385 agonist-selected, truncated variants (thermostabilized A 2A -Star2 and rant21) found also using exponential fits were ~50–500 fold higher than we determined in this study 31, 32 . Interestingly, our k off estimate and those of the other reports vary only by ~0.5–15 fold, indicating much less variability in k off for ZM 241,285 relative to k on .…”

Nanodiscs in Membrane Biochemistry and Biophysics

“…194 Possible partitioning of the ligand into the lipid bilayer of Nanodiscs was accounted for by placing empty Nanodiscs in the reference channel. Results of this work confirmed that label-free monitoring of small molecule binding kinetics of the membrane protein in Nanodiscs could be performed using SPR, suggesting a new approach to the measurement of the influence of agonistic or antagonistic ligands on the binding of G proteins or β-arrestin to the extracellular or intracellular sides of the bilayer.…”

Ligand Residence Time at G-protein–Coupled Receptors—Why We Should Take Our Time To Study It

“…Both studies demonstrate the powerful potential of this approach. The influence of lipid composition upon assay performance was recently investigated in a comparative study of the adenosine A 2A receptor employing four different reconstitution approaches (Bocquet et al, 2015). When the receptor was reconstituted in lipid nanodiscs, protein stability was enhanced and the kinetic data obtained were more similar to native receptors compared with those solubilized in detergents.…”