Journal: eLife

Article Title: Identification of an allosteric binding site on the human glycine transporter, GlyT2, for bioactive lipid analgesics

doi: 10.7554/eLife.47150

Figure Lengend Snippet: ( 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 normalised to the current elicited by glycine alone. Data represented are means ± SEM with ****p<0.001 following a one way ANOVA test. ( b–e ) All lipid inhibitors docked to GlyT2 and burrowed into an area between EL4, TM1, TM5, TM7, and TM8 during 100 ns of simulation. ( b ) The initial docked poses, viewed top down from the extracellular side of GlyT2 (grey surface). Sections of TM1, TM5, and TM7 are cut away in ( b–d ) to show the docking cavity (transparent surface). ( c ) The initial docked poses of OLLys (orange sticks), OLTrp (green sticks), OLLeu (blue sticks), and OLSer (magenta sticks) overlaid on GlyT2. ( d ) The conformations of the lipid inhibitors after 100 ns of unrestrained MD simulation, overlaid on GlyT2 from the OLLys simulation. ( e ) A close-up view of the inhibitors following 100 ns of simulation, with surrounding TM helices, including those cut away in panels ( b–d ), shown as a coloured surface. ( f–g ) Map of key regions in the extracellular allosteric site. ( f ) 3D arrangement of residues within 4 Å of OLLys (black sticks) following 100 ns of simulation. Residues have side chains shown as sticks, with side chains coloured TM1 (red), TM5 (yellow), TM7 (purple), EL4 (orange), TM8 (green). V523 is a backbone interaction. F428, R439, and L569 are >4 Å but shown for reference. ( g ) 2D representation, with residues studied via mutagenesis outlined in black.

Article Snippet: Chemical compound, drug , Oleoyl L-carnitine , Larodan , 17–1810 , .

Techniques: Inhibition, Mutagenesis

Journal: eLife

Article Title: Identification of an allosteric binding site on the human glycine transporter, GlyT2, for bioactive lipid analgesics

doi: 10.7554/eLife.47150

Figure Lengend Snippet: ( 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 cavity and interacts with the acyl tail, where there is a substantial kink adjacent to the head group. ( c ) Overlay showing snapshots of OLSer as it moves from the docked position (0 ns, green sticks) and into the cavity between TM5/7/8 of WT GlyT2 (50 ns, red sticks; 100 ns, blue sticks). I545 is shown as cyan spheres. TM5 and TM7 are transparent to show the cavity. ( d-f ) The I545L mutation (cyan spheres) sterically blocks deep insertion into the cavity. ( d ) Docked position of OLLys (magenta spheres) on the I545L mutant transporter and ( e ) following 100 ns of simulation – OLLys maintains head group interactions but the acyl tail adopts a hairpin structure to bind at a shallower cavity. ( f ) Overlay of OLSer as it leaves the docked position and cannot insert into the I545L mutant GlyT2, but instead idles above in the extracellular compartment. Coloured as in ( c ). ( g-h ) Acyl amino acids inhibit glycine transport currents of WT and mutant GlyT1 and GlyT2 transporters. Glycine transport currents were measured in the presence of lipids to generate concentration inhibition curves for ( e ). OLCarn (⊗) and ( f ) oleoyl l -Lys (□). GlyT2 WT is shown in black, GlyT1 WT is in grey, I545L (G2) is in teal, and L425I (G1) in pink.

Article Snippet: Chemical compound, drug , Oleoyl L-carnitine , Larodan , 17–1810 , .

Techniques: Binding Assay, Mutagenesis, Concentration Assay, Inhibition

Journal: eLife

Article Title: Identification of an allosteric binding site on the human glycine transporter, GlyT2, for bioactive lipid analgesics

doi: 10.7554/eLife.47150

Figure Lengend Snippet: ( 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 curves for ( a ). OLCarn (⊗),oleoyl l -Lys (□), ( b ) NOGly ( ), C18 ω8 Gly (◊), C18 ω10 Gly (⊠), ( c ) C16 ω7 Gly (∇), C16 ω11 Gly (◯) and C14 ω5 Gly ( + ). GlyT2 WT is shown in black and F428A is in orange. ( d ) Overlay of GlyT2 in the absence of inhibitor (wheat helices) with OLLys (magenta spheres) bound at 100 ns (blue helices). TM8 regions were aligned to show the relative movement of TM5. ( e ) Distances between the extracellular edges of TM5 (T442) and TM8 (S560) calculated from their Cα. ( f ) Distances between the middle of TM5 (F428) and TM8 (L569) calculated from their Cα. ( g ) Distances between the last carbon of the lipid inhibitor tail and the bottom of the extracellular allosteric pocket (Cα of V432).

Article Snippet: Chemical compound, drug , Oleoyl L-carnitine , Larodan , 17–1810 , .

Techniques: Mutagenesis, Concentration Assay, Inhibition