Role of the Second Extracellular Loop of the Adenosine A₁ Receptor on Allosteric Modulator Binding, Signaling, and Cooperativity

Abstract

Allosteric modulation of adenosine A1 receptors (A1ARs) offers a novel therapeutic approach for the treatment of numerous central and peripheral disorders; however, despite decades of research, there is a relative paucity of structural information regarding the A1AR allosteric site and mechanisms governing cooperativity with orthosteric ligands. We combined alanine-scanning mutagenesis of the A1AR second extracellular loop (ECL2) with radioligand binding and functional interaction assays to quantify effects on allosteric ligand affinity, cooperativity, and efficacy. Docking and molecular dynamics (MD) simulations were performed using an A1AR homology model based on an agonist-bound A2AAR structure. Substitution of E172ECL2 for alanine reduced the affinity of the allosteric modulators PD81723 and VCP171 for the unoccupied A1AR. Residues involved in cooperativity with the orthosteric agonist NECA were different in PD81723 and VCP171; positive cooperativity between PD81723 and NECA was reduced on alanine substitution of a number of ECL2 residues, including E170ECL2 and K173ECL2, whereas mutation of W146ECL2 and W156ECL2 decreased VCP171 cooperativity with NECA. Molecular modeling localized a likely allosteric pocket for both modulators to an extracellular vestibule that overlaps with a region used by orthosteric ligands as they transit into the canonical A1AR orthosteric site. MD simulations confirmed a key interaction between E172ECL2 and both modulators. Bound PD81723 is flanked by another residue, E170ECL2, which forms hydrogen bonds with adjacent K168ECL2 and K173ECL2. Collectively, our data suggest E172ECL2 is a key allosteric ligand-binding determinant, whereas hydrogen-bonding networks within the extracellular vestibule may facilitate the transmission of cooperativity between orthosteric and allosteric sites.

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors

“…Our results from both flexible and rigid receptor docking demonstrate that A 1 R PAMs also bind in this extracellular vestibule utilizing important interactions with E172 and K173. In the new paper by Nguyen et al, residues E172 and K173 were experimentally verified to mediate PAM activity of PD‐81,723 and VCP171 for A 1 R in agreement with our models . In addition, W156A was found to not affect allosteric ligand affinity, but did, probe‐dependently, reduce cooperativity …”

“…While this manuscript was in review, a paper by Nguyen et al was published that investigated the effects of alanine‐scanning mutagenesis and radioligand binding assays to determine their influence on allosteric ligand affinity, cooperativity, and efficacy of PAMs PD‐81,723 and VCP171 on A 1 R . The authors hypothesized that the allosteric site is most likely located within a hydrogen bonding network within the extracellular vestibule . This binding site is analogous to the allosteric binding site observed in the activated M 2 R crystal structure in complex with iperoxo and PAM LY2119620.…”

“…Presumably the closer agreement (3.6 Å) between the rigid and flexible receptor results for A 2A R compared to A 1 R were that the complexes for A 2A R were built from crystal structures in complex with the reference agonist, rather than from homology models. While this manuscript was in review, a paper by Nguyen et al was published that investigated the effects of alanine‐scanning mutagenesis and radioligand binding assays to determine their influence on allosteric ligand affinity, cooperativity, and efficacy of PAMs PD‐81,723 and VCP171 on A 1 R . The authors hypothesized that the allosteric site is most likely located within a hydrogen bonding network within the extracellular vestibule .…”

Mechanistic Insights into Specific G Protein Interactions with Adenosine Receptors Revealed by Accelerated Molecular Simulations

“…Both experimental and computational studies suggested that flexibility of the ECL2 was important for activation and allosteric modulation of the A1AR. 39,44,45 Therefore, highly flexibility of the ECL2 contributed to activation of the A1AR and receptor coupling to the G protein.…”

G-Protein-Coupled Receptor-Membrane Interactions Depend on the Receptor Activation state

“…The CHARMM36 parameter set (58) was used for the protein and POPC lipids. For agonist ADO and antagonist PSB36, the force field parameters were obtained from the CHARMM ParamChem web server (59,60). Initial energy minimization and thermalization of the A1AR system follow the same protocol as used in the previous GPCR simulations (61).…”