Nature Structural & Molecular Biology: doi: /nsmb.2307

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1 A novel locally-closed conformation of a bacterial pentameric proton-gated ion channel Marie S. Prevost*, Ludovic Sauguet*, Hugues Nury, Catherine Van Renterghem, Christèle Huon, Frederic Poitevin, Marc Baaden, Marc Delarue, Pierre-Jean Corringer Supplementary Figure 1 Pore radii and effect of DTT on the LC mutant structures. (a, b) Pore radius along the channel axis in the open and LC conformations. Radii were calculated using the program Hole (O.S. Smart, J.M. Goodfellow and B.A. Wallace. The Pore Dimensions of Gramicidin A. Biophysical Journal 65: (1993)). In the 2-22

2 mutant, the rotamer of the E-2 side chain has been changed to that of the 2-24 mutant to harmonize the curves. A cartoon representation of M2 in the Open and LC conformations is shown in b, with pore-lining residues shown as sticks. (c) X-ray structures of double-cys mutants reduced with 10 mm DTT prior to crystallogenesis return to the open conformation. In all four bridged structures, the cysteines are engaged in an intra-subunit disulfide bridge that is visible in the electron density map, except for Loop2-20. For each structure, M2, M3 and the M2M3 Loops are superimposed onto the wild-type open form (in grey). M2 and M3 are represented as cartoon and the M2M3 Loop is shown in sticks. The blue mesh around the M2M3 Loops shows the 2Fo-Fc electron density map contoured at a level of 1.5 σ. (d) DTT reduction of the Loop2-21 mutant in the crystal state versus in detergent solution. The Loop2-21 bridged mutant was reduced either in solution by preincubating the protein in 10mM DTT for 12 hours prior to crystallization or in the crystal state by soaking the crystals in 10 mm DTT for several days. When the Loop2-21 bridged mutant was reduced in solution, the crystal structure returned from a LC2 conformation to the open form. Interestingly when reduction was performed in the crystal state, the crystal structure did not adopt the open form but instead moved to a LC1 conformation, suggesting that this latter form is more stable when the disulfide bridge constraint is removed. For each structure, M2, M3 and the M2M3 Loop of LC (in green) are superimposed onto the WT open form (in grey). M2 and M3 are represented as cartoons and the M2M3 Loop is shown as sticks. The blue mesh around the M2-M3 Loops shows the 2Fo-Fc electron density map contoured at a level of 1.5 σ.

3 Supplementary Figure 2 Structural features of 2-20 and 2-22 mutants (a, b) The M2M3 loop rearrangement in the Loop2-20 bridged mutant causes a partial reorganisation of the ECD. (a) Cartoon representation of the LC1 Loop2-20 mutant (brown) superimposed to the LC1 Loop2-24 mutant (grey). In Loop2-20, the disulfide bridge between residue 33 in Loop 2 and residue 20 in the M2M3 loop causes a 5 Å translation of Loop 2 toward the centre of the channel (see enlarged view on the upper right panel). The β strands 1 and 2 that connect Loop 2 are also affected by this movement and residues 27 to 34 were found to be disordered as no interpretable electron density was observed for these residues (2Fo- Fc map contoured at a level of 1.5 σ is represented as a blue mesh). (b) B-factor comparison between the Loop2-20 and the Loop2-24 mutants. As expected, the Loop2-20 mutant displays significantly higher B factors than the Loop2-24 mutant in the region of Loop 2, suggesting that the structural distortion caused by the movement of Loop 2 propagates along the whole β sheet from Loop F to pre-β5 that is significantly less stable in the Loop2-20 mutant than in the Loop2-24 mutant. (c, d) The M2M3 Loop in the Loop2-22 bridged mutant adopts alternative conformations. (c): The Fourier difference electron density map was calculated from an omit map obtained when residues 20 to 25 were not present during the

4 final round of refinement and represented as a blue mesh contoured at a level of 3.0 σ (left) and 2.0 σ (right). At a level of 3.0 σ, the electron density allowed to build the major conformation of the M2M3 Loop that was retained in the final model (colored in green; side chain atoms are not shown). At a level of 2.0 σ the electron density reveals the presence of additional density that could be due to the presence of at least one alternative conformation of the M2M3 Loop (in grey). (d) The occurrence of alternative conformations for the M2M3 Loop of mutant Loop2-22 suggests that it is less stabilized than in the WT open and in the LC forms. In addition, the conformation of the M2M3 Loop in mutant Loop2-22 differs from that of the WT and the LC1 mutants by the loss of important hydrophobic interactions that were described previously (Figure 1). In the WT open form, the hydrophobic Leu22 and Pro23 interact with residues Phe116 and Tyr119. In the LC1 conformation Pro23 is still maintained by the residues Phe116 and Tyr119 and Leu22 is packed against Ile35 and Ile20. At the contrary, in the Loop2-22 (LC3) mutant, these key contacts are lost and the M2M3 Loop is no longer stabilized in a single conformation.

5 Supplementary Figure 3 Lipids and transmembrane cavities in the LC conformation (a) The open and LC forms of GLIC bind differentially to lipids. When the crystal structure of GLIC was solved, three partially ordered lipids per subunit were constructed in the electron density 3. One of these three lipids is located on the extracellular side of the TMD and binds to regions M3-M1-M4 and Loop 7 (homologous to the cys-loop in eukaryotes), which might therefore be partly responsible for lipid-dependent allosteric modulation 11. The phospholipids are shown in sticks mode with the protein surface shown in grey. The blue mesh shows the Fo-Fc electron density map contoured at a level of 2.5 σ, as calculated when the phospholipids were omitted in the final round of refinement. Left: The Fo-

6 Fc electron density around the lipid molecule is shown for the wild-type GLIC. The electron density was calculated using data from the PDB entry 3EAM and identifies the aliphatic and phosphate moieties of the phospholipids. The phospholipids contact the protein through both hydrophobic interactions (involving residues F121, I198, I202, L203, L206, I258, F303 and F315) and polar interactions (involving residues R118, Y194 and Y254). Right: The Fo-Fc electron density calculated for the cysteine cross-linked mutants (Loop2-20, Loop2-21, Loop2-22 and Loop2-24 ) revealed no residual electron density that could be assigned to phospholipid. When these mutants were reduced using DTT and returned back to an open form in the crystal, residual electron density in the Fo-Fc map clearly identified again the presence of phospholipids. No residual density is observed for the phospholipids in crystal-reduced Loop2-21, H11 F and E19 P. (b) top-view of the transmembrane domain of GLIC wt (left) and Loop2-24 mutant (right), with calculated intrasubunit (yellow, binding propofol and desflurane) and inter-subunit (purple) cavities, as defined in Nury et al. 10 and calculated using the program Hollow (Generating Accurate Representations of Channel and Interior Surfaces in Molecular Structures Bosco K. Ho and Franz Gruswitz. BMC Structural Biology (2008) 8:49). (c) side view of the cavities, residue 197 is depicted as sticks. In the LC1 conformation, the volume of the intra-subunit cavity is reduced by about 25% by the tilt of the Tyr197 side chain which switches from interacting with Loop 7 to hydrogen bonding Thr31 in helix M3. The inter-subunit cavity is enlarged by about 50% with the displacement of M2. The tunnel linking the intrasubunit cavity to the inter-subunit cavity (orange) is closed (right panel).

7 Supplementary Figure 4 Expression and characterization of selected mutants (a) Redox treatment on the solubilized MBP-fused GLIC mutants. After immobilization of detergent-solubilized MBP-GLIC on amylose resin, DTT (lane 2) or H 2 O 2 (lane3) is added to the buffer at 10 mm and 0.35% respectively in batch for 1h, followed by a washing step to eliminate traces of the redox agent. For lane 4, the resin is sequentially treated

8 with DTT (1h) followed by H 2 O 2 (1h). Each MBP-GLIC sample is then eluted with 20 mm maltose and submitted to a non-reducing SDS gel. The cysless control indicates that bands 2-mers correspond to an intersubunit cysteine crosslinking. These data show that only Loop2 and Loop2-20 can be significantly intersubunit cross-linked. The other constructs are in great majority intrasubunit cross-linked (do not show dimers), even after reduction and re-oxydation. Thus, for Loop2-21, 22 and 24 mutants, the H 2 O 2 oxydation favors the intrasubunit bridging as compared to the intersubunit bridging, showing reversibility of the oxydoreduction. (b) Immulolabeling of GLIC in Xenopus oocytes. Oocytes were co-injected with DNA coding for GFP alone or together with DNA coding for GLIC-HA. Four days after injection, PFA-fixed/GFP+ oocytes were submitted to immunolabeling using an anti-ha primary antibody and an anti-rabbit Cy5 coupled secondary antibody. Oocytes were then 40 µm sliced. Slices were analysed using epi-fluorescence microscopy with constant exposure times for visualisation of the HA tag. (c) Proton dose response traces for cysless and N15 C I35 C mutants. (d) Examples of traces for calculating pcmbs accessibility for the G32 C, I35 C and F36 C single mutants.

9 a b Supplementary Figure 5 Normal mode analysis and a speculative allosteric model (a) This figure describes the normal modes involved in the trajectory described in Figure 4e and f. Briefly, there is a major contribution from an M2 translation movement (orange) both before and after the transition state, the twist mode (red) contributes mostly after the transition state, and there are several local modes appearing specifically at the transition state and disappearing shortly afterwards (black). Each family of modes

10 is schematically represented in c and contains modes that were judged by eye to induce the same kind of movement. Family A (red) is the 9th mode, and is commonly referred to as the "twist" mode. Family B (magenta) corresponds to three modes: # 12, 13, 14. These modes mainly affect the upper part of the ECD. Family C (blue) corresponds to three modes: # 21, 23, 29. These modes can be seen as a "twisted twist" where the middle part of the protein rotates in phase opposition with the upper and lower parts. Family D (green) corresponds to three modes: # 24, 25, 26. These modes can be seen as a "pump" mode, expanding or compressing the protein in the vertical direction. Family E (orange) corresponds to 5 modes: # 32, 36, 37, 38, 39. These modes mainly affect the TMD, pushing the M2 helices up or down relative to the rest of the protein. Family F (cyan) corresponds to 3 modes: # 51, 52, 54. These modes affect both the TMD and ECD. Interestingly enough, they allow M1 and M3 to rotate around the pore axis. Family G (black) corresponds to 14 modes: # 56, 63, 76, 77, 99, 124, 155, 156, 172, 173, 175, 176, 194, 249. These modes affect both the ECD and TMD. For clarity, we only show the TMD, where it appears that they correspond to a radial motion of the M2M3 Loop. (b) Speculative allosteric model. Major states (Basal, Active and Desensitized) are represented together with a putative intermediate conformation (I BA or I AD ) inferred from functional analysis. M2 and M3 helices are schematically represented. We assume that the active state corresponds to the GLIC open conformation. In the absence of unambiguous structural data, M3 was drawn in dashes for the basal and desensitized conformations. In this framework, we propose that the LC conformation may correspond to I BA or I AD.

11 Supplementary Table 1 Data collection and refinement statistics Loop2-24 (Reduced) Loop2-22 (Reduced) Loop2-21 (Reduced in solution) Loop2-21 (Reduced in crystal) Loop2-20 (Reduced) Space group C2 C2 C2 C2 C2 Cell dimensions a, b, c (Å) , , , , , , , , , α, β, γ ( ) 90.0, , 90.0 Resolution (Å) * ( ) 90.0, , , 102.6, , 102.1, , , ( ) ( ) ( ) ( ) R merge * 11.6 (57.8) 6.1 (56.7) 8.4 (51.5) 7.0 (56.9) 7.3 (63.7) I / σi* 8.2 (2.1) 13.0 (2.2) 11.2 (2.3) 10.8 (2.2) 12.2 (2.2) Completeness (%)* 97.3 (98.2) 99.6 (98.1) 99.9 (99.9) 99.7 (100.0) 99.8 (99.9) Redundancy* 2.9 (2.9) 3.4 (3.4) 3.9 (3.9) 3.7 (3.7) 3.8 (3.8) Resolution (Å) No. reflections R work / R free 22.6/ / / / /21.8 Protein Ligand/Ion Water Protein # 59.9 (66.4) 78.6 (85.1) 60.4 (64.8) (108.3) 75.4 (82.2) Ligand/ion Water Bond lengths (Å) Bond angles ( ) *Values in parentheses are for the highest-resolution shell # Main values are for the mainchain atoms and values in parentheses are for the sidechain atoms

12 Supplementary Table 2. ph 50, Hill number and maximal current of engineered GLIC and GlyRα1 human mutants ph 50 /EC 50 (mm) nh I max (µa) n GLIC cysless 5.05± ± ± Loop2 5.40± ±0.15*** -4.36± ± ± ±0.15*** ± ± ±2.47* ±0.08* 2.98±0.32** -1.11±1.09*** ±0.04* 4.10±0.32*** -5.10± Loop ± ± ± Loop ± ± ±0.84*** 4 Loop ± ± ± Loop ±0.10* 1.68± ± N15 C 4.61±0.12*** 2.16±0.46* -3.28±1.36*** 6 I35 C 5.06± ±0.03*** -4.99±1.60* 3 N15 C I35 C 6.44±0.13** 1.00± ± GlyRα1 human wt 0.036± ± ±0.3 3 Loop2-22 oxidized 0.79±0.4** 1.1±0.1*** -0.2±0.06*** 3 Loop2-22 reduced 1.6±0.2** 1.7±0.3** -0.7± Loop ±0.5*** 1.5±0.05** -1.3± For this mutant, data were recorded at 48h after injection, due to cell toxicity. For the double cysteines mutants, data were obtained after 2 minutes DTT. Significance was assessed by performing a Dunnett s test using the cysless (GLIC) or wt (GlyRα1) construct as control. *: p<0.05, **: p<0.001, ***: p<0.0001

13 Supplementary Table 3. Interactions seen in GLIC WT, LC1 (H11 F) and ELIC X-ray structures at the ECD-TMD interface Loop7-M2M3 Loop7-preM1 Residue 1 Residue 1 Residue 2 Residue 2 GLIC ELIC GLIC ELIC GLIC WT GLIC LC1 ELIC (3.7) (3.5) (2.7) + 2 (2.6) + 2 (2.6) (3.6) + 3 (3.5) (2.5) (3.6) + 1 (4) (3.8) + 1 (3.5) (4)* (2.7) + 2 (2.6) (2.7) + 2 (4) + 1 (4) (3) + 1 (3.4) (3.4) + 1 (3.8) + 1 (3.8) (2.4) + 4 (2.7) + 4 (2.7) Loop7-M (3.6) + 3 (4) Loop2-preM (2.4) + 4 (2.9) Loop2-M2M3 M2M3-preM1 M2M3-M2M (3.3) + 1 (3.4) (3.3) (4) (3) (2.8)* (3.5) (3.5)* (3.4) (4.1) (3.8)* (4) (3.9)* (3.1) For each described interaction (+), inter-atom distance in Angstrom are given in brackets and nature of the interaction are indicated as following: 1 hydrophobic 2 hydrogen bond 3 cation-π interaction 4 salt bridge * inter-subunit interaction