Abstract

Serotonin (5-hydroxytryptamine; 5-HT) receptors modulate a variety of physiological processes ranging from perception, cognition and emotion to vascular and smooth muscle contraction, platelet aggregation, gastrointestinal function and reproduction. Drugs that interact with 5-HT receptors effectively treat diseases as diverse as migraine headaches, depression and obesity. Here we present four structures of a prototypical serotonin receptor—the human 5-HT2B receptor—in complex with chemically and pharmacologically diverse drugs, including methysergide, methylergonovine, lisuride and LY266097. A detailed analysis of these structures complemented by comprehensive interrogation of signaling illuminated key structural determinants essential for activation. Additional structure-guided mutagenesis experiments revealed binding pocket residues that were essential for agonist-mediated biased signaling and β-arrestin2 translocation. Given the importance of 5-HT receptors for a large number of therapeutic indications, insights derived from these studies should accelerate the design of safer and more effective medications.

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Acknowledgements

We thank R. Axel (Columbia University) for the HTLA cells expressing TEV-fused β-arrestin2 and R. Fischetti and the staff of APS GM/CA for assistance in the development and use of the minibeam and beam time at GM/CA-CAT beamline 23-ID at the Advanced Photon Source, which is supported by National Cancer Institute grant Y1-CO-1020 and National Institute of General Medical Sciences grant Y1-GM-1104. Use of the Advanced Photon Source was supported by the Office of Science of the US Department of Energy. This work was supported by US National Institutes of Health (NIH) grants R01MH61887 (B.L.R.), R01NS100930 (J.J.), U19MH82441 (J.J. and B.L.R.) and F31-NS093917 (R.H.J.O.), the NIMH Psychoactive Drug Screening Program Contract (B.L.R.) and the Michael Hooker Distinguished Chair of Pharmacology (B.L.R.).

Author information

Author notes

    • John D. McCorvy

    Present address: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA

    • Daniel Wacker

    Present address: Department of Pharmacological Sciences and Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    • Sheng Wang

    Present address: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

  1. These authors contributed equally: John D. McCorvy, Daniel Wacker, Sheng Wang.

Affiliations

  1. National Institute of Mental Health Psychoactive Drug Screening Program, Department of Pharmacology and Division of Chemical Biology and Medicinal Chemistry, University of North Carolina Chapel Hill Medical School, Chapel Hill, NC, USA

    • John D. McCorvy
    • , Daniel Wacker
    • , Sheng Wang
    • , Bemnat Agegnehu
    • , Katherine Lansu
    • , Alexandra R. Tribo
    • , Reid H. J. Olsen
    • , Tao Che
    •  & Bryan L. Roth
  2. Center for Chemical Biology and Drug Discovery, Department of Pharmacological Sciences and Department of Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    • Jing Liu
    •  & Jian Jin

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Contributions

J.D.M. conceived the project, designed the mutant constructs and experiments, performed pharmacological assays, analyzed the data and wrote the manuscript; D.W. expressed protein, purified the receptor, optimized crystallization conditions, grew crystals for data collection, collected and processed diffraction data, supervised structure determination and assisted with preparing the manuscript; S.W. expressed protein, purified the receptor, optimized crystallization conditions, grew crystals for data collection, collected and processed diffraction data and assisted with preparing the manuscript; B.A. expressed protein, purified receptor, optimized crystallization conditions and grew crystals for data collection; J.L. designed and synthesized LY266097 analogs and performed analytical chemical analysis; K.L. assisted with performing PI hydrolysis signaling studies and analyzed the data; A.R.T. assisted with performing β-arrestin recruitment experiments; R.H.J.O. assisted with performing BRET experiments; T.C. assisted with binding studies; J.J. supervised ligand synthesis and edited the manuscript; B.L.R. was responsible for the overall project strategy and management and edited the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to John D. McCorvy or Bryan L. Roth.

Integrated supplementary information

  1. Supplementary Figure 1 Activation mechanisms and 5-HT2BR–methylergonovine structure.

    a, Additional 5-HT2BR ergoline SAR showing Gq-mediated calcium flux by N(1)-H-containing ergolines, LSD (red, EC50 = 26 nM, Emax = 82%) and ergotamine (ERG, black, EC50 = 334 nM, Emax = 85%) and antagonism by N(1)-alkyl ergolines, including LY215840, which contains N(1)-isopropyl (blue, IC50 = 2.0 nM). Data are expressed as percent 5-HT response and represent means ± s.e.m. from two independent experiments (n = 2) performed in triplicate. b, Alignment of the 5-HT2BR ERG (gray), LSD (purple) and methylergonovine (light blue) crystal structures reveals slight differences in positioning of the indole N(1)-H with respect to TM5. c, 5-HT2BR–methylergonovine structure FoFc omit map of ligand (left), 2FoFc regular map of ligand (middle) and binding pocket residues (right). d, Surface expression measured by ELISA reveals similar expression levels of the T1403.37 and A2255.46 mutants as for wild-type 5-HT2BR. Data represent means ± s.e.m. from quadruplicate replicates from two independent experiments (n = 2). e, FoFc omit density map of residues T1403.37 and L3627.35 in the 5-HT2BR–methylergonovine structure. f, 5-HT Gq-mediated calcium flux agonist potency at T140A3.37 (red, EC50 = 73 nM) and T140V3.37 (blue, EC50 = 93 nM) compared to T140S3.37 (green, EC50 = 5.7 nM) and wild-type 5-HT2BR (black, EC50 = 5.2 nM). Data are expressed as fold-over-basal and represent means ± s.e.m. from two independent experiments (n= 2) performed in triplicate. g, 5-HT Gq-mediated calcium flux agonist potency at A225S5.46 (red, EC50 = 1.4 nM) and A225G5.46 (blue, EC50 = 1.5 nM) as compared to wild-type 5-HT2BR (black, EC50 = 3.2 nM). Data are expressed as fold-over-basal and represent means ± s.e.m. from two independent experiments (n= 2) performed in triplicate.

  2. Supplementary Figure 2 Activation mechanism and 5-HT2BR(A225G5.46)–methysergide structure.

    a, 5-HT2BR Gq-mediated PI hydrolysis showing similar basal levels of IP accumulation and Gq agonist activity by methysergide for A225G5.46 (green, EC50 = 0.7 nM) compared to no measured agonist activity for wild-type 5-HT2BR (black). Data are expressed as counts per minute (CPM) of [3H]IP and represent means ± s.e.m. from three independent experiments (n= 3) performed in duplicate. b, 5-HT2BR(A225G5.46)–methysergide structure FoFc omit map of ligand (left), 2FoFc regular map of ligand (middle) and binding pocket residues (right). c, FoFc omit density map of residues T1403.37 and L3627.35 in the 5-HT2BR(A225G5.46)–methysergide structure. d, Alignment of the structure for the β2AR–ICI-118,551 complex (blue) with the nanobody-stabilized active state of β2AR in complex with epinephrine (green) indicates that the methyl of ICI-118,551 precludes inward TM5 movement.

  3. Supplementary Figure 3 5-HT2BR–lisuride structure and OBP mutant L3627.35 surface expression.

    a, 5-HT2BR–lisuride structure FoFc omit map of ligand (left), 2FoFc regular map of ligand (middle) and binding pocket residues (right). b, FoFc omit density map of residues T1403.37 and L3627.35 in the structure of lisuride-bound 5-HT2BR. c, Alignment of the structures for the 5-HT2BR–LSD and 5-HT2BR–lisuride complexes reveals similar positioning of the indole N(1)-H with respect to TM5. d, Surface expression measured by ELISA reveals similar expression levels of L3627.35 mutants as for wild-type 5-HT2BR. Data represent means ± s.e.m. from quadruplicate replicates from two independent experiments (n= 2).

  4. Supplementary Figure 4 Divergent function by OBP versus EBP mutations.

    a, Left, 5-HT Gq-mediated calcium flux comparing T140A3.37 (red, EC50 = 130 nM) to wild-type 5-HT2BR (black, EC50 = 5.2 nM). Right, 5-HT β-arrestin2 recruitment comparing T140A3.37 (red, EC50 = 178 nM) to wild-type 5-HT2BR (black, EC50 = 3.6 nM). b, Left, 5-HT Gq-mediated calcium flux comparing A225G5.46 (green, EC50 = 1.5 nM) to wild-type 5-HT2BR (black, EC50 = 2.2 nM). Right, 5-HT β-arrestin2 recruitment comparing A225G5.46 (green, EC50 = 4.6 nM) to wild-type 5-HT2BR (black, EC50 = 3.6 nM). Data in a and b are expressed as fold-over-basal and represent means ± s.e.m. from two independent experiments (n= 2) performed in triplicate. c, LSD β-arrestin2 recruitment for the L362A7.35 (red), L362N7.35 (orange), L362Y7.35 (blue) and L362F7.35 (green) 5-HT2BR mutants. Data are expressed as luminescent counts per second (LCPS), indicating β-arrestin2 recruitment as measured by Tango, and represent means ± s.e.m. from two independent experiments (n= 2) performed in triplicate. d, Mutant 5-HT2BR L362F7.35 Gq-mediated PI hydrolysis by 5-HT (black, EC50 = 1.8 nM) and lisuride (blue, EC50 = 0.65 nM). Data are expressed as counts per minute (CPM) of [3H]IP and represent means ± s.e.m. from two independent experiments (n= 2) performed in duplicate. e, Mutant 5-HT2BR L362F7.35 Gq/ γ1 dissociation as measured by BRET2 showing 5-HT (black, EC50 = 1.3 nM) and lisuride (blue, EC50 = 1.2 nM) Gq agonist activity. Data are represented as means ± s.e.m. from two independent experiments (n= 2) and are expressed as NET BRET ratio (Methods). f, Mutant 5-HT2BR L362F7.35 LSD β-arrestin2 recruitment as measured by BRET1. Data are represented as means ± s.e.m. from two independent experiments (n= 2) and are expressed as NET BRET ratio (Methods).

  5. Supplementary Figure 5 5-HT2BR structure and function by LY266097.

    a,5-HT2BR–LY266097 structure FoFc omit map of ligand (left), 2FoFc regular map of ligand (middle) and binding pocket residues (right). b, FoFc omit density map of residues T1403.37 and L3627.35 in the 5-HT2BR–LY266097 structure. c, LY266097 5-HT2BR Gq-mediated PI hydrolysis (EC50 = 3.6 nM, Emax = 52%). Data are expressed as percent 5-HT response and represent means ± s.e.m. from three independent experiments (n= 3) performed in duplicate. d, LY266097 β-arrestin2 recruitment as measured by BRET1 over 5, 30, 60 and 120 min. Data represent means ± s.e.m. from two independent experiments (n= 2) performed in duplicate and are expressed as percent 5-HT NET BRET ratio response. e, β-arrestin2 recruitment as measured by Tango comparing agonist activity of 5-HT (black, EC50 = 11 nM) to LY266097 (blue, closed circles), where LY266097 exhibits β-arrestin2 recruitment antagonism (blue, open circles, IC50 = 2.2 nM) of 5-HT. Data represent means ± s.e.m. from two independent experiments (n= 2) performed in triplicate and are expressed as percent 5-HT response.

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https://doi.org/10.1038/s41594-018-0116-7