The E2.65A mutation disrupts dynamic binding poses of SB269652 at the dopamine D2 and D3 receptors

The dopamine D2 and D3 receptors (D2R and D3R) are important targets for antipsychotics and for the treatment of drug abuse. SB269652, a bitopic ligand that simultaneously binds both the orthosteric binding site (OBS) and a secondary binding pocket (SBP) in both D2R and D3R, was found to be a negative allosteric modulator. Previous studies identified Glu2.65 in the SBP to be a key determinant of both the affinity of SB269652 and the magnitude of its cooperativity with orthosteric ligands, as the E2.65A mutation decreased both of these parameters. However, the proposed hydrogen bond (H-bond) between Glu2.65 and the indole moiety of SB269652 is not a strong interaction, and a structure activity relationship study of SB269652 indicates that this H-bond may not be the only element that determines its allosteric properties. To understand the structural basis of the observed phenotype of E2.65A, we carried out molecular dynamics simulations with a cumulative length of ~77 μs of D2R and D3R wild-type and their E2.65A mutants bound to SB269652. In combination with Markov state model analysis and by characterizing the equilibria of ligand binding modes in different conditions, we found that in both D2R and D3R, whereas the tetrahydroisoquinoline moiety of SB269652 is stably bound in the OBS, the indole-2-carboxamide moiety is dynamic and only intermittently forms H-bonds with Glu2.65. Our results also indicate that the E2.65A mutation significantly affects the overall shape and size of the SBP, as well as the conformation of the N terminus. Thus, our findings suggest that the key role of Glu2.65 in mediating the allosteric properties of SB269652 extends beyond a direct interaction with SB269652, and provide structural insights for rational design of SB269652 derivatives that may retain its allosteric properties.Author summary: G protein-coupled receptors (GPCRs) are targets of more than 25% of prescription drugs on the market. Due to their critical roles in human physiology, competitive modulation of these receptors has been found to be associated with many undesired side effects. Allosteric modulation holds the promise of retaining normal receptor function and improving selectivity. However, the underlying molecular mechanisms of the allosteric modulation of GPCRs have remained largely uncharted. The dopamine D2-like receptors have been implicated in voluntary movement, reward, sleep, learning, and memory. Based on previous experimental findings, we computationally characterized the binding of a negative allosteric modulator of dopamine D2 and D3 receptors, and revealed the dynamic binding mode of this modulator in a secondary binding pocket (SBP) of the receptors. Our results highlight the key role of a Glu in mediating the allosteric properties of the modulator by shaping the dynamically formed SBP, and shed light on rational design and optimization of allosteric modulators of GPCRs.