Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site

  1. Marijke Brams
  2. Cedric Govaerts
  3. Kumiko Kambara
  4. Kerry L Price
  5. Radovan Spurny
  6. Anant Gharpure
  7. Els Pardon
  8. Genevieve L Evans
  9. Daniel Bertrand
  10. Sarah CR Lummis
  11. Ryan E Hibbs
  12. Jan Steyaert
  13. Chris Ulens  Is a corresponding author
  1. Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, Faculty of Medicine, KU Leuven, Belgium
  2. Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université libre de Bruxelles, Belgium
  3. HiQscreen, Switzerland
  4. Department of Biochemistry, University of Cambridge, United Kingdom
  5. Department of Neuroscience, University of Texas Southwestern Medical Center, United States
  6. Department of Biophysics, University of Texas Southwestern Medical Center, United States
  7. Structural Biology Brussels, Vrije Universiteit Brussel, Belgium
  8. VIB-VUB Center for Structural Biology, VIB, Belgium
5 figures, 1 table and 4 additional files

Figures

Figure 1 with 3 supplements
Nanobodies active as allosteric modulators and structures bound to the ELIC channel.

(a, b) Electrophysiological recordings of ELIC activated by the agonist GABA and in the presence of increasing concentrations of PAM-Nb (a, green) or NAM-Nb (b, red). The curve represents a fit to …

Figure 1—source data 1

Electrophysiological recordings of ELIC with PAM-Nb and NAM-Nb.

https://cdn.elifesciences.org/articles/51511/elife-51511-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
The effect of PAM-Nb and NAM-Nb on the GABA concentration-activation curve.

(a) An example control experiment with two consecutive applications of a series of GABA concentrations (5–40 mM). ELIC is prone to desensitization, but this effect can be minimized by excluding GABA …

Figure 1—figure supplement 1—source data 1

ELIC concentration-activation curves in the presence of PAM-Nb and NAM-Nb.

https://cdn.elifesciences.org/articles/51511/elife-51511-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Simulated annealing omit map for PAM-Nb ELIC structure.

The white mesh shows a simulated annealing omit map calculated in PHENIX and contoured at a sigma level of 1.0 around a 12 Å sphere zoom of the ELIC-Nb interface. In the omit calculation an omit box …

Figure 1—figure supplement 3
Simulated annealing omit map for NAM-Nb ELIC structure.

The white mesh shows a simulated annealing omit map calculated in PHENIX and contoured at a sigma level of 1.0 around a 12 Å sphere zoom of the ELIC-Nb interface. In the omit calculation, an omit …

Figure 2 with 3 supplements
Detailed nanobody interaction sites in ELIC and channel pore analysis.

(a) A detailed view of the interaction between PAM-Nb (green) and a single ELIC subunit (blue). The binding site for PAM-Nb overlaps with a known allosteric binding site, in a related pLGIC, for a …

Figure 2—figure supplement 1
Detailed atomic interactions between PAM-Nb and ELIC, compared to those between CU2017 (a NAM molecule) and α7-AChBP (Delbart et al., 2018).

(a–b) The cartoon shows that the binding site of the PAM-Nb (green) in ELIC (blue) (a) is homologous to the CU2017 (yellow spheres) site in α7-AChBP (cyan) (b). For clarity, only residues at the …

Figure 2—figure supplement 1—source data 1

List of amino acid interactions in the PAM-Nb bound ELIC structure versus CU2017 bound alpha7-AChBP structure.

https://cdn.elifesciences.org/articles/51511/elife-51511-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Detailed atomic interactions between NAM-Nb and ELIC, compared to those between flurazepam (a PAM molecule) and ELIC (Spurny et al., 2012).

(a) The cartoon shows that the binding site of the NAM-Nb (red) is adjacent to the flurazepam site (yellow spheres) in the vestibule of ELIC (orange). For clarity, only residues at the receptor site …

Figure 2—figure supplement 2—source data 1

List of amino acid interactions in the NAM-Nb bound ELIC structure versus flurazepam bound ELIC structure.

https://cdn.elifesciences.org/articles/51511/elife-51511-fig2-figsupp2-data1-v2.xlsx
Figure 2—figure supplement 3
Analysis of pore radius profiles through lateral fenestrations located at subunit interfaces in ELIC and the β3 GABAAR (Miller and Aricescu, 2014).

(a) Pore radius profiles extending into lateral fenestrations (green, magenta, blue) at subunit interfaces or the extracellular vestibule entrance (amber) in the NAM-Nb bound structure. (b) Pore …

Figure 3 with 1 supplement
Distinct conformations of the vestibule site in pentameric ligand-gated ion channels.

(a–c) Yellow ribbon representation of a single subunit ligand binding domain. Part of the vestibule site (shown in blue cartoon), called the Ω-loop, adopts three distinct conformations in different …

Figure 3—figure supplement 1
B-factor analysis of the Ω-loop compared to other regions within each structure.

The vestibule site is depicted in the ‘putty’ cartoon presentation. Yellow-red regions indicate example structures with higher average B-factors per residue compared to cyan-blue regions. B-factors …

Allosteric modulation of the 5-HT3A receptor through chemical modification of engineered cysteines in the vestibule site.

(a) Example traces of agonist-evoked channel responses of the L151C 5-HT3AR mutant before and after modification with MTSEA-biotin show potentiation after cysteine modification. (b) Location of …

Figure 4—source data 1

Electrophysiological recordings of 5-HT3R mutants before and after treatment with MTSEA-biotin.

https://cdn.elifesciences.org/articles/51511/elife-51511-fig4-data1-v2.xlsx
Author response image 1

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
GeneErwinia ligand-gated ion channel (ELIC)Synthetic gene from GenscriptUniProt
P0C7B7
GeneHuman 5-hydroxytryptamine receptor 3A (5-HT3A R)John Peters laboratory (Belelli et al., 1995)GenBank NM_000869
Cell lineC43 E. coli strainLucigen#60446
Cell lineWK6 E. coli strainZell and Fritz, 1987
Cell lineTG1 E. coli strainLucigen#60502
AntibodyPAM-NbNanobody obtained by immunizing an adult llama glama with recombinant ELIC protein. PAM-Nb was used at 1–30 μM concentration in electrophysiology experiments. In protein crystals the PAM-Nb concentration is > 1 mM.
AntibodyNAM-NbNanobody obtained by immunizing an adult llama glama with recombinant ELIC protein. NAM-Nb was used at 0.03–3 μM concentration in electrophysiology experiments. In protein crystals the NAM-Nb concentration is > 1 mM.
Recombinant DNA reagentpGEM-HELiman et al. (1992)
Recombinant DNA reagentpMESy4GenBank KF415192
Sequence-based reagentP111C oligonucleotide forward and reverse5’-caccaagttgtccatcTGcacggacagcatctgg-3’
5’-ccagatgctgtccgtgCAgatggacaacttggtg- 3’
Sequence-based reagentT112C oligonucleotide forward and reverse5’-caagttgtccatccccTGCgacagcatctgggtcc-3’
5’-ggacccagatgctgtcGCAggggatggacaacttg-3’
Sequence-based reagentN147C oligonucleotide forward and reverse5’-caaggcgaagttcagTGctacaagccccttcagg-3’
5’-cctgaaggggcttgtagCActgaacttcgccttg-3’
Sequence-based reagentK149C oligonucleotide forward and reverse5’-ggcgaagttcagaactacTGCccccttcaggtggtga-3’
5’- tcaccacctgaaggggGCAgtagttctgaacttcgcc-3’
Sequence-based reagentL151C oligonucleotide forward and reverse5’- gttcagaactacaagcccTGtcaggtggtgactgc-3’
5’-gcagtcaccacctgaCAgggcttgtagttctgaac-3’
Sequence-based reagentY86C oligonucleotide forward and reverse5’- ctggtaccggcagtGctggactgatgag-3’
5’-ctcatcagtccagCactgccggtaccag-3’
Sequence-based reagentF125C oligonucleotide forward and reverse5’-ggacattctcatcaatgagtGcgtggatgtggg-3’
5’-cccacatccacgCactcattgatgagaatgtcc-3’
Sequence-based reagentPrimer for nanobody library generation
CALL001
Pardon et al. (2014)
5′-GTCCTGGCTGCTCTTCTACAAGG-3′
Sequence-based reagentPrimer for nanobody library generation
CALL002
Pardon et al. (2014)
5′-GGT ACGTGCTGTTGAACTGTTCC-3′
Sequence-based reagentPrimer for nanobody library generation
VHH-Back
Pardon et al. (2014)
5′-GATGTGCAGCTGCAG GAGTCTGGRGGAGG-3′(PstI)
Sequence-based reagentPrimer for nanobody library generation
VHH-For
Pardon et al. (2014)
5′-CTAGTGCGGCCGCTGG AGACGGTGACCTGGGT-3′(Eco91I)
Sequence-based reagentPrimer for nanobody library analysis
MP57
Pardon et al. (2014)
5′-TTATGCTTCCGGCTC GTATG-3′
Peptide, recombinant proteinPrimer for nanobody library analysis
GIII
Pardon et al. (2014)
5′-CCACAGACAGCCCTCATAG-3′
Commercial assay or kitmMessage mMachine T7 Transcription kitThermoFisher Scientific#AM1344
Commercial assay or kitNi Sepharose 6 Fast Flow resinGE Healthcare#17531802
Commercial assay or kitSuperdex 75 10/300 GL columnGE Healthcare# 17-5174-01
Commercial assay or kitSuperdex 200 Increase 10/300 GLGE Healthcare# 28990944
Commercial assay or kitQuikChange Site-Directed Mutagenesis KitAgilent# 200518
Chemical compound, drug5-HT creatinine sulphateSigma-Aldrich#S2805
Chemical compound, drugMTSEA-biotinBiotium#90064
Chemical compound, drugGABASigma-Aldrich#A2129
Chemical compound, drugE. coli total lipid extractAvanti Polar Lipids#100500P
Chemical compound, drugAnagrade n-undecyl-β-D-maltoside (UDM)Anatrace#U300
Software, algorithmGraphPad Prism Software v4.03RRID:SCR_002798
Software, algorithmCCP4Winn et al., 2011
Software, algorithmSTARANISOTickle et al., 2018 http://staraniso.globalphasing.org
Software, algorithmXDSKabsch, 2010
Software, algorithmCootEmsley et al. (2010)
Software, algorithmBusterSmart et al. (2012)
Software, algorithmPDB-REDOJoosten et al. (2014)
Software, algorithmMolProbityChen et al. (2010)
Software, algorithmPyMOL v2.3.0SchrödingerRRID:SCR_000305
Software, algorithmHOLESmart et al. (1996)
Software, algorithmCAVERJurcik et al. (2018)
Software, algorithmPHENIXAdams et al. (2010)
Software, algorithmHiClampMulti Channel Systems

Additional files

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