Nature Structural & Molecular Biology: doi: /nsmb.2548

Transcription

1 Supplementary Figure 1. Structure of GltPhout. (a) Stereo view of a slice through a single GltPhout protomer shown in stick representation along with 2Fo-Fc and anomalous difference

2 electron maps. The former is colored blue and contoured at 1 σ, and the latter is black and contoured at 5 σ. Both maps were calculated for Å resolution range and were subject to three fold real space averaging. The Cα atoms of the cross-linked cysteines are emphasized as yellow spheres. (b) Stereo view of the structural superposition of single WT Glt Ph (PDB accession code 2nwl) and Glt Ph out protomers shown in ribbon representation. The superposition was generated using residues , which correspond to the structurally conserved region between outward and inward facing states. The trimerization and transport domains are colored orange and blue for the wild type and brown and black for Glt Ph out, respectively. The substrate and the cross-linking Hg 2+ ion are emphasized as spheres, and the cysteines are shown in stick representation.

3 Supplementary Figure 2. RH421 dye senses coupled Na + /Asp binding to Glt Ph and TBA inhibits transport catalyzed by Glt Ph. (a) Emission spectra of RH421 in a buffer containing in mm 10 HEPES/KOH ph 7.4, 99 KCl (black line); after addition of 0.2 mm DDM and 1 µm Glt Ph -WT (blue line); and after further addition of 10 mm NaCl and 1 mm Asp (green line). Fluorescence was excited at 532 nm. (b) Proteo-liposomes, containing WT Glt Ph at protein to lipid ratio of 1:50 were loaded with buffer containing 300 mm choline chloride and assayed in the uptake buffer containing 100 mm NaCl, 200 mm choline chloride, 100 nm 3 H-Asp and the indicated amounts of TBA (open circles) or DL-TBOA (solid circles) for 10 minutes. Averages of three measurements with standard errors are shown.

4 Supplementary Figure 3. TBA and Asp bind in a competitive manner. Binding isotherms for Glt Ph out (blue) and Glt Ph in (red) at 25 C. (a) TBA titrations in the presence of 100 mm NaCl. The solid black lines through the data are fits with the following parameters: K D = 3.8 and 1.5 µm, ΔH = -2.6 and -8.7 kcal/mol and n = 1.0 and 1.0 for Glt Ph out and Glt Ph in, respectively. (b) Asp titrations in the presence of 0.6 mm TBA and 30 mm NaCl. The solid black lines through the data are fits with the following parameters: K D = 0.13 and 0.43 µm, ΔH = and -8.3 kcal/mol and n = 0.9 and 0.5 for Glt Ph out and Glt Ph in, respectively.

5 Supplementary Figure 4. Na+ binding in the presence of coupled ligands. Na+ titrations derived from fluorescence experiments using GltPhout (a, blue), GltPhin (b, red) and the WT (c, black) at 25 C. Left panels are Na+ titrations in the presence of 1 (solid circles), 0.1 (open circles) and 0.01 (solid triangles) mm Asp. Right panels are Na+ titrations in the presence of 10 (solid circles), 1 (open circles) and 0.1 (solid triangles) mm TBA. The data were fitted to Hill equations yielding average Hill coefficients, nhill as indicated on the graphs.

6 Supplementary Figure 5. Asp bind in 1M Na+ and determination of the binding state functions. (a) Binding isotherms for GltPhout (blue, left) and GltPhin (red, right) at 25 C in the presence of 1M NaCl. Upper panels, the solid colored lines are the heat powers developed during the titration. Lower panels, the black solid lines through the data are fits with the following parameters: ΔH = and kcal/mol and n = 0.7 and 0.9 for GltPhout and GltPhin, respectively. The KD values could not be calculated accurately due to the lack of experimental points in the transition regions of the plots. (b) Schematic representation of the reactions for which thermodynamic parameters were obtained experimentally (solid lines) or calculated (dashed lines). Left panel, H0s and CPs were determined experimentally for Asp binding under three conditions: in the presence of [Na+] < 60 mm (reaction 1), in the presence of [Na+] = 1 M (reaction 2) and in the presence of [TBA] = 0.6 mm and [Na+] = 30 mm (reaction 3). The H0s and CPs for the binding of 2 Na+ ions and consequent L-TBA binding were calculated as differences between those for reactions 1 and 2, and for reactions 2 and 3, respectively. Right panel, G0s for 3Na/Asp (reaction 1) and 2Na/L-TBA (reaction 3) binding were determined from the linear extrapolations in Fig. 2c and G0 of 2 Na+ binding alone (reaction 2) measured

7 directly. The G 0 s of Asp and L-TBA binding to the transporters pre-bound to 2 Na + ions were calculated as differences between those for reactions 1 and 2, and for reactions 3 and 2, respectively. G 0 of L-TBA replacement by Asp was calculated as a difference between that of reactions 1 and 3.

8 out in Supplementary Figure 6. DL-TBOA binding. Shown are data for Glt Ph (blue) and Glt Ph (red). a, ITC data for DL-TBOA titrations in the presence of 100 mm NaCl at 25 C. The solid black lines through the data are fits with the following parameters: K D = 2.6 and 16.0 µm, ΔH = -9.8 and -8.9 kcal/mol and n = 1.4 and 1.2 for Glt out Ph and Glt in Ph, respectively. b, Temperature dependencies of DL-TBOA binding enthalpies obtained at 200 mm NaCl. Straight line fits to the data yielded ΔC P estimates of -148 and -334 cal mol -1 deg -1 for Glt out Ph and Glt in Ph, respectively.

9 Supplementary Table 1: Thermodynamic parameters of binding reactions Hg 2+ binding 1 mm Na + K D, nm ΔH, kcal/mol Glt Ph -L66C/S300C Glt Ph -K55C/A364C mm Na +, 0.2 mm Asp Glt Ph -L66C/S300C Glt Ph -K55C/A364C # Binding parameters were obtained by fitting data in Figure 1, c and d to independent binding sites model. n is the number of the binding sites. n # Competitive binding of TBA and Asp. Glt Ph out Glt Ph in ΔG Asp ΔG TBA, ΔG TBA to Asp, ΔG calc TBA to Asp, kcal/mol kcal/mol kcal/mol kcal/mol Glt Ph out Glt Ph in ΔH 0 Asp, kcal/mol ΔH 0 TBA, kcal/mol ΔH 0 TBA to Asp, kcal/mol ΔH 0 TBA to Asp calc, kcal/mol ± ± ± ± ± ± G Asp, G TBA, H 0 Asp and H 0 TBA are Asp and TBA binding free energies and standard enthalpies, respectively. The Gs were measured by ITC in the presence of 30 mm Na +. The H 0 s are averages of at least three ITC experiments performed at Na + concentrations between 10 and 100 mm (Fig. 2c). G TBA to Asp, G calc TBA to Asp, H TBA to Asp and H calc TBA to Asp are the experimental and calculated free energies and enthalpies of TBA replacement by Asp, respectively. The experimental G TBA to Asp and H 0 TBA to Asp were obtained by ITC in the presence of 0.6 mm TBA and 30 mm Na +. The enthalpies are averages of three experiments. calc G TBA to Asp was calculated using the following competition equation: K D, calc =K D,Asp (1+[TBA]/K D,TBA ), where K D, Asp and K D, TBA correspond to G Asp and G TBA, respectively. The competition equation is valid when concentration of TBA is significantly higher than that of the protein. In our experiments, TBA to protein molar ratio was at least 15. H 0 TBA to Asp calc are the differences between the binding enthalpies of Asp and TBA.

10 SUPPLEMENTARY NOTE The standard free energy of Asp and Na + binding. The coupled binding reaction is: T + Asp + "# Na + $ T# Asp# Na " +, (S1) where T represents the transporter, and α is the number of ions coupled to Asp binding. The total free energy of this reaction is: "G Total = "G 0 + RT lna TAspNa # RT lna Asp # $% RT lna Na # RT lna T, (S2) where G 0 is the standard free energy, defined as the free energy of the reaction at 25 C, 1 atm pressure and 1 M concentration of the reactants, and the a T, a TAspNa+, a Asp and a Na are the activities of the free transporter, the bound transporter, Asp and Na +, respectively. At a given Na + activity, G Total = 0 and a T = a TAspNa+ when the activity of Asp equals to apparent dissociation constant K D,app. RT lnk D,app = "G 0 # $% RT ln a Na (S3) Considering that RTlnK D,app = G app, where G app is the apparent free energy of Asp binding, and RTlna Na = µ Na, where µ Na is the chemical potential of the ion, we obtain the final expression of equation (1): "G app = "G 0 # $% µ Na Calculations of the thermodynamic parameters. The thermodynamic parameters for 3Na + /Asp binding to Glt Ph out and Glt Ph in, including H, G and Cp were measured directly by ITC. However, the thermodynamic parameters of Na + ions biding could not be determined by ITC

11 because the affinity for Na + is too low for direct measurements. Instead, H and Cp could be assessed from T + Asp + 3Na + " 1 T# Asp# Na 3 + T + 2Na + " 2 T# Na Asp + Na + " 3 T# Asp# Na 3 + (S4) In the above scheme, H and Cp values for reactions (1) and (3) were experimentally determined (experimentally accessible reactions 1 and 2, respectively, in Supplementary Fig. 7a), and the corresponding values for reaction (4) were calculated as differences between (1) and (3): H 2 = H 1 - H 3 and Cp 2 = Cp 1 - Cp 3 (S5) G for reaction (2) was measured directly using fluorescence-based assay. The Asp affinity to Na + -saturated transporter, reaction (3), is too high to measure by either ITC or the fluorescence assay, and G 3 of this reaction was instead calculated as (Supplementary Fig. 7b): G 3 = G 1 - G 2 (S6) Similarly, H and Cp for TBA binding could not be determined by ITC because of insufficient heats of binding. Gs were still accessible from the fluorescence-based affinity measurements. Instead, H and Cp were determined via a two-step process: the transporters were first saturated with TBA, and then titrated with Asp. In the scheme below, the experimentally observed reactions are (1) and (5) (experimentally accessible reactions 1 and 3, respectively, in Supplementary Fig. 7a), while reaction (4) cannot be characterized directly: T + Asp + 3Na + " 1 T# Asp# Na 3 + T + TBA + 2Na + " 4 T# TBA# Na Asp + Na + " 5 T# Asp# Na TBA (S7)

12 assuming Na + stoichiometry of 2 and 3 for TBA and Asp binding, respectively. The values of the thermodynamic parameters for reaction (4) were calculated as differences between corresponding values of reactions (1) and (5). Temperature dependence of the binding free energy. The temperature dependences of the free energies were calculated using the thermodynamic parameters either measured or calculated at T ref = 298 K as described above, and the corresponding Cp values based on the following standard thermodynamic expressions: "H(T) = "H(T ref ) + "Cp(T # T ref ) (S8) "S(T) = "S(T ref ) + "Cp# ln T T ref (S9) "G(T) = "H(T) # T$ "S(T) (S10)