Structural Connection between Activation Microswitch and Allosteric Sodium Site in GPCR Signaling

Abstract

Sodium ions are endogenous allosteric modulators of many G-protein-coupled receptors (GPCRs). Mutation of key residues in the sodium binding motif causes a striking effect on G-protein signaling. We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A adenosine receptor, D52N and S91A. Both structures present an overall active-like conformation; however, the variants show key changes in the activation motif NPxxY. Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G-protein signaling and enhanced thermal stability. These structures, signaling data, and thermal stability analysis with a panel of pharmacological ligands provide a basis for understanding the role of the sodium-coordinating residues on stability and G-protein signaling. Utilizing the DN variant is a promising method for stabilizing class A GPCRs to accelerate structural efforts and drug discovery.

Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions

“…These studies show that, upon the addition of a full agonist, A2aR activation follows outward movements of TM5 and TM6 (including rotation in the latter), an inward shift of the intracellular part of TM7, and a vertical translation of TM3 [42,[61][62][63][64]. Also, A2aR has received a lot of attention regarding ligand binding, lipid allosteric modulation and its activation process in MD simulations [44,[65][66][67][68][69][70][71][72][73][74][75][76][77][78] partly because it is a receptor that has been crystallized in three distinct conformational states: inactive (in the presence of an antagonist or inverse agonist) [29,63,[79][80][81][82][83][84][85][86][87][88][89][90][91], intermediate (in the presence of an agonist) [92][93][94][95], and active (in the presence of an agonist plus modified or native stimulatory G proteins) [24,25] (S1 Table).…”

The mutational landscape of human olfactory G protein-coupled receptors

“…It is characterized by the presence of a negative ionizable residue in the conserved (N/S)LxxxD 2.50 motif, which is involved in the GPCR activation mechanism through allosteric modulation mediated by ionic species [52] ( Figure 8A, C and E). Replacement of the conserved D 2.50 would impair the coordination of modulating ions due to the loss of the negatively ionizable center [53].…”

7-Transmembrane Helical (7TMH) Proteins: Pseudo-Symmetry and Conformational Plasticity

“…Its symmetry related, highly conserved residue D 2.50 on TM2 (in protodomain 1) does not bind to the ligand. In fact, D2.50 binds to Na + that has been shown to correlate with the functional state of the receptors (White et al, 2018). It can be symmetrically paired with either F5.44 or W5.48, both highly conserved.…”

“…Just below the ligand binding area, the highly conserved class A GPCR residues S3.39 and N/S7.45 (or S7.46) match symmetrically across the protodomains. They are Na + binding residues (White et al, 2018). Below the Na + binding area, when one looks at helix-helix contacts that change upon activation (Cvicek et al, 2016;Venkatakrishnan et al, 2016), TM3 and TM7 form contacts between residues at positions 3.…”