Structures of the 5-HT2A receptor in complex with the antipsychotics risperidone and zotepine

Kimura, Kanako; Asada, Hidetsugu; Inoue, Asuka; Kadji, Francois Marie Ngako; Im, Dohyun; Mori, Chihiro; Arakawa, T.; Hirata, Kunio; Nomura, Yoshihiro; Aoki, Junken; Iwata, So; Shimamura, Tatsuro

doi:10.1038/s41594-018-0180-z

Cited by 158 publications

(187 citation statements)

References 39 publications

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“…An atomistic insight into those conformational states of 5‐HT 2A R are of supreme importance for a better understanding of the receptor responses to different ligands. The marked increase in the crystallography of membrane proteins led to the elucidation of the structures of several members of the GPCR family, including the inverse agonist‐bound 5‐HT 2A R . However, in most available structures (Figure S1), the receptor sequence is reengineered to reduce instability and to favor crystallization process by truncating various segments, including regions of N and C‐termini along with the intracellular loop 3 (ICL3), despite their utmost importance in the context of physiological functions .…”

Section: Introductionmentioning

confidence: 99%

Comprehensive structural modeling and preparation of human 5‐HT_2A G‐protein coupled receptor in functionally active form

et al. 2019

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The serotonin 2A receptor (5-HT 2A R) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT 2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT 2A R has marked distinction with ligand-bound conformations of 5-HT 2 subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT 2A R by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier. K E Y W O R D S 5-HT 2A GPCR, baculoviral expression system, homology modeling, molecular dynamics simulation, protein over-expression and purification

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Section: Introductionmentioning

confidence: 99%

Comprehensive structural modeling and preparation of human 5‐HT_2A G‐protein coupled receptor in functionally active form

et al. 2019

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“…Nevertheless, risperidone retains all other contacts in the OBS. In the recently reported 5-HT2AR/risperidone structure (PDB: 6A93) 20 , risperidone has a very similar pose in the OBS as that in the D2R structure, occupying the Ile 3.40 sub-pocket as well. However, on the extracellular side of the OBS, EL2 in the 5-HT2AR/risperidone complex is in an extended conformation and the EL2 residue Leu228 EL2.51 contacting risperidone aligns to Ile183 EL2.51 of D2R, whereas the conserved Trp141 EL1.50 does not interact with risperidone in the 5-HT2AR.…”

Section: El2 and El1 Dynamicsmentioning

confidence: 96%

“…However, on the extracellular side of the OBS, EL2 in the 5-HT2AR/risperidone complex is in an extended conformation and the EL2 residue Leu228 EL2.51 contacting risperidone aligns to Ile183 EL2.51 of D2R, whereas the conserved Trp141 EL1.50 does not interact with risperidone in the 5-HT2AR. It is tempting to speculate the EL2 and EL1 dynamics we observe in the D2R/risperidone simulations represents a more comprehensive picture, as the divergent interactions shown in the extracellular loops of the 5-HT2AR/risperidone and D2R/risperidone structures may not result from differences in recognizing the ligand but rather two different static snapshots due a variety of differences in the crystallographic conditions (Note risperidone has similarly high affinities for both D2R and 5HT2AR 10,20 ). Interestingly, in one of our long MD trajectories of the D2R/risperidone complex, EL2 evolved into a conformation that has a helical N-terminal portion and an extended C-terminal portion ( Supplementary Movie 4 and Supplementary Fig.…”

Section: El2 and El1 Dynamicsmentioning

confidence: 99%

“…2). Intriguingly, in the recently reported crystal structure of the serotonin 2A receptor (5-HT2AR) in complex with zotepine (PDB: 6A94) 20 , the benzothiepin moiety of zotepine also occupies the Ile 3.40 sub-pocket, but this results in noticeable differences in the nearby residues compared to the 5-HT2AR/risperidone structure (PDB: 6A93) 20 . Specifically, the sidechain of the thermostabilizing mutation S162 3.39 K, which occupies the Na + binding site, moves further away from Ile163 3.40 in the presence of the bound zotepine compared to that in the presence of risperidone.…”

Section: Mutagenesis Of Ile122 340 and The Sensitivity To Na +mentioning

confidence: 99%

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Distinct antagonist-bound inactive states underlie the divergence in the structures of the dopamine D2 and D3 receptors

Lane

Verma

et al. 2019

Preprint

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Understanding how crystal structures reflect the range of possible G protein-coupled receptor (GPCR) states is critical for rational drug discovery (RDD). Combining computational simulations with mutagenesis and binding studies, we find that the structure of the dopamine D2 receptor (D2R)/risperidone complex captures an inactive receptor conformation that accommodates some but not all antagonist scaffolds. Indeed, we find that eticlopride binds D2R in a configuration very similar to that seen in the D3R structure, in a pose that is incompatible with the D2R/risperidone structure. Moreover, our simulations reveal that extracellular loops 1 and 2 (EL1 and EL2) are highly dynamic, with spontaneous transitions of EL2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results highlight previously unappreciated conformational diversity and dynamics in the inactive state of a GPCR with potential functional implications. These findings are also of paramount importance for RDD as limiting a virtual screen to one state will miss relevant ligands.

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“…While the structures of serotonin receptors have been well studied , only recently have experimental structures of MT receptors been obtained , providing the missing piece to understanding MT/5‐HT polypharmacology and structural templates for rational design of tool compounds selective for melatonin receptor subtypes and more efficient drugs including safer sleeping aids.…”

Section: Introductionmentioning

confidence: 99%

Structural insights into melatonin receptors

2019

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The long-anticipated high-resolution structures of the human melatonin G protein-coupled receptors MT 1 and MT 2 , involved in establishing and maintaining circadian rhythm, were obtained in complex with two melatonin analogs and two approved anti-insomnia and antidepression drugs using X-ray free-electron laser serial femtosecond crystallography. The structures shed light on the overall conformation and unusual structural features of melatonin receptors, as well as their ligand binding sites and the melatonergic pharmacophore, thereby providing insights into receptor subtype selectivity. The structures revealed an occluded orthosteric ligand binding site with a membrane-buried channel for ligand entry in both receptors, and an additional putative ligand entry path in MT 2 from the extracellular side. This unexpected ligand entry mode contributes to facilitating the high specificity with which melatonin receptors bind their cognate ligand and exclude structurally similar molecules such as serotonin, the biosynthetic precursor of melatonin. Finally, the MT 2 structure allowed accurate mapping of type 2 diabetes-related single-nucleotide polymorphisms, where a clustering of residues in helices I and II on the protein-membrane interface was observed which could potentially influence receptor oligomerization. The role of receptor oligomerization is further discussed in light of the differential interaction of MT 1 and MT 2 with GPR50, a regulatory melatonin coreceptor. The melatonin receptor structures will facilitate design of selective tool compounds to further dissect the specific physiological function of each receptor subtype as well as provide a structural basis for next-generation sleeping aids and other drugs targeting these receptors with higher specificity and fewer side effects.

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Structures of the 5-HT2A receptor in complex with the antipsychotics risperidone and zotepine

Cited by 158 publications

References 39 publications

Comprehensive structural modeling and preparation of human 5‐HT_2A G‐protein coupled receptor in functionally active form

Comprehensive structural modeling and preparation of human 5‐HT_2A G‐protein coupled receptor in functionally active form

Distinct antagonist-bound inactive states underlie the divergence in the structures of the dopamine D2 and D3 receptors

Structural insights into melatonin receptors

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Structures of the 5-HT2A receptor in complex with the antipsychotics risperidone and zotepine

Cited by 158 publications

References 39 publications

Comprehensive structural modeling and preparation of human 5‐HT2A G‐protein coupled receptor in functionally active form

Comprehensive structural modeling and preparation of human 5‐HT2A G‐protein coupled receptor in functionally active form

Distinct antagonist-bound inactive states underlie the divergence in the structures of the dopamine D2 and D3 receptors

Structural insights into melatonin receptors

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Product

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Comprehensive structural modeling and preparation of human 5‐HT_2A G‐protein coupled receptor in functionally active form

Comprehensive structural modeling and preparation of human 5‐HT_2A G‐protein coupled receptor in functionally active form