Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site
- Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, Faculty of Medicine, KU Leuven, Belgium
- Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université libre de Bruxelles, Belgium
- HiQscreen, Switzerland
- Department of Biochemistry, University of Cambridge, United Kingdom
- Department of Neuroscience, University of Texas Southwestern Medical Center, United States
- Department of Biophysics, University of Texas Southwestern Medical Center, United States
- Structural Biology Brussels, Vrije Universiteit Brussel, Belgium
- VIB-VUB Center for Structural Biology, VIB, Belgium
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 …
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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 …
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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 …
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Figure 2—source data 1
Analysis of pore radius profiles.
- https://cdn.elifesciences.org/articles/51511/elife-51511-fig2-data1-v2.xlsx
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 …
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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 …
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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 …
Figure 4
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 …
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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 | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene | Erwinia ligand-gated ion channel (ELIC) | Synthetic gene from Genscript | UniProt P0C7B7 | |
| Gene | Human 5-hydroxytryptamine receptor 3A (5-HT3A R) | John Peters laboratory (Belelli et al., 1995) | GenBank NM_000869 | |
| Cell line | C43 E. coli strain | Lucigen | #60446 | |
| Cell line | WK6 E. coli strain | Zell and Fritz, 1987 | ||
| Cell line | TG1 E. coli strain | Lucigen | #60502 | |
| Antibody | PAM-Nb | Nanobody 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. | ||
| Antibody | NAM-Nb | Nanobody 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 reagent | pGEM-HE | Liman et al. (1992) | ||
| Recombinant DNA reagent | pMESy4 | GenBank KF415192 | ||
| Sequence-based reagent | P111C oligonucleotide forward and reverse | 5’-caccaagttgtccatcTGcacggacagcatctgg-3’ 5’-ccagatgctgtccgtgCAgatggacaacttggtg- 3’ | ||
| Sequence-based reagent | T112C oligonucleotide forward and reverse | 5’-caagttgtccatccccTGCgacagcatctgggtcc-3’ 5’-ggacccagatgctgtcGCAggggatggacaacttg-3’ | ||
| Sequence-based reagent | N147C oligonucleotide forward and reverse | 5’-caaggcgaagttcagTGctacaagccccttcagg-3’ 5’-cctgaaggggcttgtagCActgaacttcgccttg-3’ | ||
| Sequence-based reagent | K149C oligonucleotide forward and reverse | 5’-ggcgaagttcagaactacTGCccccttcaggtggtga-3’ 5’- tcaccacctgaaggggGCAgtagttctgaacttcgcc-3’ | ||
| Sequence-based reagent | L151C oligonucleotide forward and reverse | 5’- gttcagaactacaagcccTGtcaggtggtgactgc-3’ 5’-gcagtcaccacctgaCAgggcttgtagttctgaac-3’ | ||
| Sequence-based reagent | Y86C oligonucleotide forward and reverse | 5’- ctggtaccggcagtGctggactgatgag-3’ 5’-ctcatcagtccagCactgccggtaccag-3’ | ||
| Sequence-based reagent | F125C oligonucleotide forward and reverse | 5’-ggacattctcatcaatgagtGcgtggatgtggg-3’ 5’-cccacatccacgCactcattgatgagaatgtcc-3’ | ||
| Sequence-based reagent | Primer for nanobody library generation CALL001 | Pardon et al. (2014) | 5′-GTCCTGGCTGCTCTTCTACAAGG-3′ | |
| Sequence-based reagent | Primer for nanobody library generation CALL002 | Pardon et al. (2014) | 5′-GGT ACGTGCTGTTGAACTGTTCC-3′ | |
| Sequence-based reagent | Primer for nanobody library generation VHH-Back | Pardon et al. (2014) | 5′-GATGTGCAGCTGCAG GAGTCTGGRGGAGG-3′(PstI) | |
| Sequence-based reagent | Primer for nanobody library generation VHH-For | Pardon et al. (2014) | 5′-CTAGTGCGGCCGCTGG AGACGGTGACCTGGGT-3′(Eco91I) | |
| Sequence-based reagent | Primer for nanobody library analysis MP57 | Pardon et al. (2014) | 5′-TTATGCTTCCGGCTC GTATG-3′ | |
| Peptide, recombinant protein | Primer for nanobody library analysis GIII | Pardon et al. (2014) | 5′-CCACAGACAGCCCTCATAG-3′ | |
| Commercial assay or kit | mMessage mMachine T7 Transcription kit | ThermoFisher Scientific | #AM1344 | |
| Commercial assay or kit | Ni Sepharose 6 Fast Flow resin | GE Healthcare | #17531802 | |
| Commercial assay or kit | Superdex 75 10/300 GL column | GE Healthcare | # 17-5174-01 | |
| Commercial assay or kit | Superdex 200 Increase 10/300 GL | GE Healthcare | # 28990944 | |
| Commercial assay or kit | QuikChange Site-Directed Mutagenesis Kit | Agilent | # 200518 | |
| Chemical compound, drug | 5-HT creatinine sulphate | Sigma-Aldrich | #S2805 | |
| Chemical compound, drug | MTSEA-biotin | Biotium | #90064 | |
| Chemical compound, drug | GABA | Sigma-Aldrich | #A2129 | |
| Chemical compound, drug | E. coli total lipid extract | Avanti Polar Lipids | #100500P | |
| Chemical compound, drug | Anagrade n-undecyl-β-D-maltoside (UDM) | Anatrace | #U300 | |
| Software, algorithm | GraphPad Prism Software v4.03 | RRID:SCR_002798 | ||
| Software, algorithm | CCP4 | Winn et al., 2011 | ||
| Software, algorithm | STARANISO | Tickle et al., 2018 http://staraniso.globalphasing.org | ||
| Software, algorithm | XDS | Kabsch, 2010 | ||
| Software, algorithm | Coot | Emsley et al. (2010) | ||
| Software, algorithm | Buster | Smart et al. (2012) | ||
| Software, algorithm | PDB-REDO | Joosten et al. (2014) | ||
| Software, algorithm | MolProbity | Chen et al. (2010) | ||
| Software, algorithm | PyMOL v2.3.0 | Schrödinger | RRID:SCR_000305 | |
| Software, algorithm | HOLE | Smart et al. (1996) | ||
| Software, algorithm | CAVER | Jurcik et al. (2018) | ||
| Software, algorithm | PHENIX | Adams et al. (2010) | ||
| Software, algorithm | HiClamp | Multi Channel Systems |
Additional files
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Supplementary file 1
Crystallographic and refinement statistics.
- https://cdn.elifesciences.org/articles/51511/elife-51511-supp1-v2.docx
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Supplementary file 2
Structures used for structure-based superposition and Ω-loop analysis.
- https://cdn.elifesciences.org/articles/51511/elife-51511-supp2-v2.docx
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Supplementary file 3
Effects of MTSEA-biotin (MB) application on WT and mutant 5-HT3R.
- https://cdn.elifesciences.org/articles/51511/elife-51511-supp3-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/51511/elife-51511-transrepform-v2.docx
