Identification of an allosteric binding site on the human glycine transporter, GlyT2, for bioactive lipid analgesics
- University of Sydney, Australia
- The Australian National University, Australia
- University of Technology Sydney, Australia
Figures
Figure 1 with 10 supplements
Defining the bioactive lipid binding site.
(a) Inhibition of GlyT2 by 1 µM oleoyl L-carnitine. Transporters were WT or with mutations to the vestibule allosteric site (VAS) or adjacent to EL4. The reduced glycine transport currents were …
Figure 1—figure supplement 1
Protonation states of OLLys (orange), OLTrp (green) OLLeu (blue), and OLSer (magenta) used in the docking and molecular dynamics simulations.
https://doi.org/10.7554/eLife.47150.003
Figure 1—figure supplement 2
Location of mutated residues on GlyT2.
(a) Reverse face (b) Top down. Residues which were mutated are shown in licorice representation and coloured red (oxygen), blue (nitrogen), and either magenta or cyan (carbon) for residues in the …
Figure 1—figure supplement 3
Sequence alignment of LeuT, hSERT, dDAT, hDAT, GlyT2, and GlyT1.
Conserved residues are coloured grey. I545, R531, and K532 are indicated with coloured green boxes. Residues tested in the vestibule allosteric site are coloured magenta, and mutated residues chosen …
Figure 1—figure supplement 4
The search space for the docking to the GlyT2 homology model (grey ribbons) was defined as a rectangular box (green/red/blue shaded walls) near mutated residues on EL4, TM5, TM7 and TM8.
https://doi.org/10.7554/eLife.47150.006
Figure 1—figure supplement 5
Resulting poses from docking to the GlyT2 homology model (grey ribbons), with EL4 highlighted in orange.
For each inhibitor, the headgroup docked in a similar location, with relative differences in the spatial orientation of the tail. Poses were classified as (1) the head inserted into extracellular …
Figure 1—figure supplement 6
After 100 ns of simulation, GlyT2 retained a similar overall conformation when the lipid inhibitors were present (GlyT2 with OLLys - blue) or absent (GlyT2 WT - wheat), as shown.
(a) In plane of the membrane and (b) Top down. Extracellular loops EL2 and EL6 displayed the highest increase in root-mean-square fluctuation (RMSF) between the unbound and inhibitor-bound …
Figure 1—figure supplement 7
The root-mean-square deviation (RMSD) of GlyT2 with no lipid inhibitor bound or when the lipid inhibitor is bound with tail inserted into extracellular pocket and directed towards TM5.
The RMSD was calculated for protein backbone.
Figure 1—figure supplement 8
The root-mean-square deviation (RMSD) of GlyT2 when the lipid inhibitor is bound with the head inserted into extracellular pocket.
The RMSD was calculated for protein backbone.
Figure 1—figure supplement 9
The root-mean-square deviation (RMSD) of GlyT2 when the lipid inhibitor is bound with tail inserted into extracellular pocket and directed towards EL4.
The RMSD was calculated for protein backbone.
Figure 1—figure supplement 10
The root-mean-square fluctuation (RMSF) of GlyT2 diverged in specific, local regions of GlyT2 when no lipid inhibitor is bound.
(a), or when the lipid inhibitor is bound with the head inserted into extracellular pocket (b), tail inserted into extracellular pocket and directed towards EL4 (c), tail inserted into extracellular …
Figure 2
I545 facilitates lipid inhibitor binding to the extracellular allosteric site.
(a) Docked position of OLLys (magenta spheres) where the acyl tail is folded at the double bond, and neighbours EL4. (b). Throughout the simulation I545 (cyan spheres) faces towards the binding …
Figure 3
Y550 coordinates the amino acid head group.
(a) Snapshot showing how the distance between C3 on the acyl-amino acids (OLLys shown here, in orange sticks) and Cα on Y550 was calculated. (b) The OLLys/OLLeu C3 (orange and blue lines, …
Figure 4
W563 and R439 act as ‘gates’ to stabilise the acyl amino acids in their binding cavity.
(a-d) Snapshots of interactions between bioactive lipids (spheres) and W563 and R439 (cyan sticks) during the simulation. GlyT2 helices are coloured EL4 (orange), TM5 (yellow), TM7 (purple), and TM8 …
Figure 5
Residues in TM5 and TM8 mediate inter-helical contacts and shape the acyl tail binding cavity.
(a-c) Acyl amino acids inhibit glycine transport currents of F428A mutant GlyT2 transporters. Glycine transport currents were measured in the presence of lipids to generate concentration inhibition …
Figure 6
Bioactive lipid inhibitors bind to GlyT2 at an extracellular allosteric site, separate from the ‘classical’ central substrate and vestibule allosteric binding sites.
OLLys (orange spheres) is bound to GlyT2 (100 ns). The binding location of s-citalopram at the substrate (maroon spheres) and vestibule allosteric (blue spheres) sites is superimposed from the …
Videos
Video 1
OLSer binding to WT GlyT2.
https://doi.org/10.7554/eLife.47150.014
Video 2
OLSer binding to I545L GlyT2.
https://doi.org/10.7554/eLife.47150.015Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (human) | GlyT2a WT | UniProtKB - Q9Y345 SLC6A5 | Morrow et al., 1998 | |
| Gene (human) | GlyT21b WT | UniProtKB - P48067 SLC6A9 | ||
| Biological sample (female Xenopus laevis) | Oocytes | Nasco, Wisconsin, USA | RRID:XEP_Xla100 | |
| Recombinant DNA reagent | pOTV | Krieg PA, Melton DA (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12:7057–7070 | ||
| Sequence-based reagent | Oligonucleotide primers | Sigma Aldrich (Sydney, Australia) | Primer designs were generated using https://nebasechanger.neb.com/ | |
| Commercial assay or kit | Q5 site-directed mutagenesis kit | New England Biolabs (Genesearch), Arundel, Australia | NEB.E0552S: | |
| Commercial assay or kit | mMessagemMachine T7 RNA polymerase | Ambion (Texas, USA) | AM1344 | |
| Commercial assay or kit | GeneJet Plasmid Mini Prep Kit | Thermo Fisher Scientific | K0503 | |
| Chemical compound, drug | N-arachidonyl glycine | Sapphire Biosciences | 90051 | |
| Chemical compound, drug | N-oleoyl glycine | Sapphire Biosciences | 90269 | |
| Chemical compounds, drugs | acyl-amino acids | Mostyn et al., 2019 | ||
| Chemical compound, drug | Oleoyl L-carnitine | Larodan | 17–1810 | |
| Chemical compound, drug | Colleganse A | Sigma Aldrich (Sydney, Australia) | 11088793001 | |
| Chemical compound, drug | Sodium bicarbonate | Sigma Aldrich (Sydney, Australia) | S5761-500G | |
| Chemical compound, drug | Tricaine | Sigma Aldrich (Sydney, Australia) | A5040-100G | |
| Chemical compound, drug | Sodium Pyruvate | Sigma Aldrich (Sydney, Australia) | P2256-25G | |
| Chemical compound, drug | Theophylline | Sigma Aldrich (Sydney, Australia) | T1633-50G | |
| Chemical compound, drug | Ampicillin | Astral Scientific | BIOAB0028-20g | |
| Chemical compound, drug | Gentamycin | Sigma Aldrich (Sydney, Australia) | G1272-10ML | |
| Chemical compound, drug | Tetracycline hydrochloride | Sigma Aldrich (Sydney, Australia) | T7660-25G | |
| Chemical compound, drug | Glycine | Sigma | 410225–50G | |
| Software, algorithm | Labchart | ADInstruments, Sydney, Australia | ||
| Software, algorithm | Pymol | Schrodinger LLC | ||
| Software, algorithm | GraphPad Prism 7 | GraphPad Software, San Diego, CA | ||
| Software, algorithm | Gromacs 2016.1 | DOI: 10.1016/j.softx.2015.06.001 | ||
| Software, algorithm | visual molecular dynamics | DOI: 10.1016/0263-7855(96)00018-5 | ||
| Software, algorithm | Autodock vina | DOI: 10.1002/jcc.21334 | ||
| Software, algorithm | gnuplot | DOI: 10.1002/jae.885 | ||
| Other | Drummond Nanoinject | Drummond Scientific Co., Broomall, PA, USA | ||
| Other | Powerlab 2/20 chart recorder | ADInstruments, Sydney, Australia | ||
| Other | Geneclamp 500 amplifier | Axon Instruments, Foster City, CA, USA |
Additional files
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Supplementary file 1
IC50 values and % max. inhibition for mutant glycine transporters.
- https://doi.org/10.7554/eLife.47150.020
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Supplementary file 2
Summary of acyl amino acids, their activity on WT transporters, and their effects in MD simulations and electrophysiological recordings of mutant transporters.
- https://doi.org/10.7554/eLife.47150.021
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Supplementary file 3
EC50 values for glycine transport of WT and mutant GlyT2 and GlyT1 transporters.
- https://doi.org/10.7554/eLife.47150.022
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Supplementary file 4
Percentage of the total simulation time in which residues are in contact with the lipid inhibitors.
- https://doi.org/10.7554/eLife.47150.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.47150.024
