Biochemical and Structural Insights on the Poplar Tau Glutathione Transferase GSTU19 and 20 Paralogs Binding Flavonoids

Glutathione transferases (GSTs) constitute a widespread superfamily of enzymes notably involved in xenobiotic detoxification and/or in specialized metabolism. Populus trichocarpa genome (V4.1 assembly, Phytozome 13) consists of 74 genes coding for full-length GSTs and ten likely pseudogenes. These GSTs are divided into 11 classes, in which the tau class (GSTU) is the most abundant with 54 isoforms. PtGSTU19 and 20, two paralogs sharing more than 91% sequence identity (95% of sequence similarity), would have diverged from a common ancestor of P. trichocarpa and P. yatungensis species. These enzymes display the distinctive glutathione (GSH)-conjugation and peroxidase activities against model substrates. The resolution of the crystal structures of these proteins revealed significant structural differences despite their high sequence identity. PtGSTU20 has a well-defined deep pocket in the active site whereas the bottom of this pocket is disordered in PtGSTU19. In a screen of potential ligands, we were able to identify an interaction with flavonoids. Some of them, previously identified in poplar (chrysin, galangin, and pinocembrin), inhibited GSH-conjugation activity of both enzymes with a more pronounced effect on PtGSTU20. The crystal structures of PtGSTU20 complexed with these molecules provide evidence for their potential involvement in flavonoid transport in P. trichocarpa.


Figure S2 Phylogenetic tree of poplar GSTUs.
Primary sequences of poplar GSTUs were retrieved from Populus trichocarpa genome (v4.1 assembly) and aligned with Clustal Omega implemented in Seaview software (Gouy et al., 2010). The phylogenetic tree was then built using the BioNJ software after curing alignment from hypervariable regions with GBlocks software (Seaview). The robustness of the tree was assessed by the bootstrap method (500 replications). PtGSTU19 and 20 isoforms are highlighted in red and conserved motif in active site is indicated. The scale corresponds to 0.1 substitution per site.

Figure S3. Purification of PtGSTU19 and PtGSTU20 isoforms.
A. Coomassie blue-stained sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of total (T), soluble (S), insoluble (I) protein fraction from E. coli BL21 (DE3) pSBET propagating PtGSTU19 (left) or PtGSTU20 (right)-producing plasmid grown in absence or presence of 0.1 mM IPTG. Fractions collected during ammonium sulfate precipitation step (0%-40% and 40%-80%), size-exclusion chromatography (SEC) and ion-exchange chromatography (IEC) were also presented. MM: molecular mass marker. B. Purified PtGSTU19 (left) and PtGSTU20 (right) proteins (300 µg in 300 µl of lysis buffer) were analyzed by SEC-MALS using an analytical Superdex200 10/300 column connected to a multi-angle light scattering (MALS) detector (miniDAWN TREOS, Wyatt technology) and a refractometer (T-rEX, Wyatt Technology). Data were processed using Astra 7 software (Wyatt Technology). C. Molecular masses of purified PtGSTU19 and 20 deduced from primary sequences and determined by mass spectrometry using a Bruker microTOF-Q spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an Apollo II electrospray ionization source with an ion funnel and operated in the negative ion mode. Ability to bind GS-sepharose is also indicated. The oxidized glutathione form and both conformations of GSH are shown as sticks in PtGSTU19 and 20, respectively. The carbon atoms are colored green and blue in PtGSTU19 and 20, respectively. All non-carbon atoms are colored according to their types (red, oxygen; blue nitrogen; yellow, sulfur).

Figure S5. Binding of Glutathione in the G-site of PtGSTU20.
Only one conformation of GSH is shown for clarity. GSH is represented in sticks as the residues surrounding it. Intermolecular contacts are depicted as dashed lines. The N-and C-terminal domains are colored orange and magenta, respectively and GSH is colored green. Non-carbon atoms are colored according to their types (red, oxygen; blue nitrogen; yellow, sulfur).

Figure S7. Views highlighting the difference between the apo-form and the GSH-complex of both PtGSTU19 and 20.
A. Apo form of PtGSTU19. Nearly two thirds of the helix 6 was found in two conformations (ConfA in blue and ConfB in yellow). Tyr160 and W161 residues were in quite distinct orientations in both conformations. B. PtGSTU19 in complex with GSOH. Only the ConfB was observed. The side chain of Tyr160 points towards the glutathione molecule. C. Apo form of PtGSTU20. Only ConfA was observed. Cys160 replaces Tyr160 of PtGSTU19. In the apo form, region 34 to 51 (helix α2 and its upstream and downstream loops) was not visible in the electron density maps. D. PtGSTU20 in complex with GSH. The region 34 to 51 (helix α2 and its upstream and downstream loops) was visible in the electron density maps and adopts the same conformation as observed in PtGSTU19.

Figure S9. Effects of different compounds from the chemical library on the thermostability of PtGSTU19 and PtGSTU20 isoforms.
Thermostability of PtGSTU19 (blue bars) and 20 (red bars) isoforms has been analyzed by using 20 μM of protein with or without 100 µM of chemical compounds diluted in 8% DMSO (Table S2). The denaturation temperature difference (ΔTd) corresponds to the difference between the denaturation temperature of the protein in presence of a potential ligand and a reference assay in which the potential ligand is replaced by the equivalent DMSO concentration. Figure S10. Stereoviews of the 2mFo-DFc maps in the putative H-site of PtGSTU20 in U20 MOR , U20 GAL , U20 BAI and U20 PIN crystal structures.
The 2mFo-DFc maps were calculated using Buster (Smart et al., 2012) and the figures were generated by Coot (Emsley and Cowtan, 2004. Each map was contoured at 1.0 σ level where σ is the standard deviation of the map.