Globally Monitoring Allosteric Coupling in the A2A Adenosine Receptor by NMR in Solution

Abstract

In human G protein‐coupled receptors (GPCRs), drug binding near the extracellular region drives conformational changes over 30 Å away at the intracellular surface, leading to complex formation with partner proteins that initiate signaling. GPCR crystal structures have revealed the need for deeper understanding of conformational dynamics to describe mechanisms of allosteric coupling between the extracellular and intracellular surfaces.Using nuclear magnetic resonance (NMR) in solution, we provide a structural basis for allosteric coupling in the human A2A adenosine receptor (A2AAR), a validated drug target for Parkinson's disease and cancer co‐immunotherapies. Production of A2AAR in Pichia pastoris allowed stable‐isotope labeling with 2H,15N, and 13C, yielding reproducible high‐quality NMR correlation spectra. Assignment of endogenous tryptophans and glycines provided numerous probes distributed over the receptor intracelluar surface, extracellular surface, and transmembrane domain, enabling a global characterization of A2AAR responses to variable efficacy of bound drugs. The “toggle switch” Trp 2466.48 was especially sensitive to variable drug efficacy due to the changes in relative orientation of the nearby aromatic ring of Phe 2426.44 in the highly conserved Pro5.50‐Ile3.40‐Phe6.44 (P‐I‐F) activation motif, providing a highly responsive probe of changes in structure and dynamics upon agonist binding. The novel NMR probes also enabled us to observe ligand efficacy‐related local conformational polymorphisms near the intracellular surface.The distributed NMR probes were utilized to monitor the impact of a key mutation in the A2AAR allosteric center at Asp522.50. Amino acid replacement of Asp522.50 leads to immediate loss of intracellular signaling. In NMR studies, this mutation caused striking changes in conformational polymorphisms at the intracellular surface but did not alter response to drug efficacy at the extracellular surface. We correlated changes at the intracellular surface to structural rearrangements at Trp 2466.48 and the P‐I‐F motif, demonstrating an interplay between the allosteric center and Trp 2466.48, and providing novel insights into a signaling mechanism that may be applicable to a wide range of class A GPCRs.Support or Funding InformationAmerican Cancer Society (MTE)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.