Modeling and Mutational Analysis of a Putative Sodium-Binding Pocket on the Dopamine D2 Receptor

SB269,652 has been described as the first negative allosteric modulator (NAM) of the dopamine D2 receptor (D2R), however, the binding mode and allosteric mechanism of action of this ligand remain incompletely understood. SB269,652 comprises an orthosteric, primary pharmacophore and a secondary (or allosteric) pharmacophore joined by a hydrophilic cyclohexyl linker and is known to form corresponding interactions with the orthosteric binding site (OBS) and the secondary binding pocket (SBP) in the D2R. Here, we observed a surprisingly low potency of SB269,652 to negatively modulate the D2R-mediated activation of G protein-coupled inward-rectifier potassium channels (GIRK) and decided to perform a more detailed investigation of the interaction between dopamine and SB269,652. The results indicated that the SB269,652 inhibitory potency is increased 6.6-fold upon ligand pre-incubation, compared to the simultaneous co-application with dopamine. Mutagenesis experiments implicated both S193 in the OBS and E95 in the SBP in the effect of pre-application. The present findings extend previous knowledge about how SB269,652 competes with dopamine at the D2R and may be useful for the development of novel D2R ligands, such as antipsychotic drug candidates.

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Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1516579113 ◽

2016 ◽

Vol 113 (13) ◽

pp. 3539-3544 ◽

Cited By ~ 25

Author(s):

Yun-Min Sung ◽

Angela D. Wilkins ◽

Gustavo J. Rodriguez ◽

Theodore G. Wensel ◽

Olivier Lichtarge

Keyword(s):

Amino Acid ◽

Dopamine D2 Receptor ◽

D2 Receptor ◽

Binding Pocket ◽

Structural Basis ◽

Functional Interactions ◽

Dopamine D2 ◽

Allosteric Communication ◽

Close Sequence ◽

Coupling Potential

The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues.

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