Structural Biology

Biased in the Absence of Sodium

Sci. Signal.  18 Feb 2014:
Vol. 7, Issue 313, pp. ec45
DOI: 10.1126/scisignal.2005184

The application of x-ray crystallography techniques to study membrane-bound receptors, such as G protein–coupled receptors (GPCRs), can reveal molecular and atomic mechanisms of receptor regulation, which can aid in the development of highly specific therapeutics. Sodium ions allosterically modulate the ligand-binding affinity of opioid receptors (ORs). Fenalti et al. crystallized the δ opioid receptor (δ-OR) complexed with the antagonist naltrindole, which revealed a sodium ion that stabilized a closed conformation of the intracellular loop 3 (ILC3). This sodium ion-dependent conformation was mediated by residues conserved with other classes of opioid receptors but was distinct from a different mechanism that stabilizes a closed conformation of the ILC3 in GPCRs of other families. The coordination of the sodium ion established a molecular connection from the allosteric sodium-binding site to the orthosteric ligand-binding site, which could explain the altered affinity of some ligands in the presence of physiological concentrations of NaCl. In addition, the sodium ion and eight associated water molecules established extensive intrahelical hydrogen-bonding interactions, which could affect the activation of the receptor. To test the functional effects of the sodium ion, heterologously expressed sodium coordinating point mutants were assessed for their ability to couple to either G protein or β-arrestin signaling pathways or for ligand binding. Mutation of Asn131 produced receptors with increased ligand-independent coupling to the β-arrestin pathway and an increased affinity for the enkephalin agonist DADLE. Sodium exhibited a reduced effect on the N131V mutant compared with the wild-type receptor, and this mutant exhibited some, albeit reduced, coupling to Gαi, whereas the N131A mutant was insensitive to sodium and failed to stimulate Gαi signaling. Mutation of another residue in the sodium binding site, Asp95, also abolished the sodium effect on ligand binding and converted apparent antagonists into agonists that were coupled to the β-arrestin pathway. Thus, the crystal structure revealed a mechanism by which sodium ions influence not only ligand affinity but also coupling efficacy, which could aid in the development of functionally biased drugs.

G. Fenalti, P. M. Giguere, V. Katritch, X.-P. Huang, A. A. Thompson, V. Cherezov, B. L. Roth, R. C. Stevens, Molecular control of δ-opioid receptor signalling. Nature 506, 191–196 (2014). [PubMed]