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1 doi:1.138/nature1737 Supplementary Table 1 variant Description FSEC - 2B12 a FSEC - 6A1 a K d (leucine) c Leucine uptake e K (wild-type like) K (Y18F) K (TS) K (TSY) K288A mutant, lipid facing side chain in TM7 Tyr 18 Phe (TM3) mutation in K Weakens substrate binding to S1 site Na2 site mutation - Thr 354 Val and Ser 355 Ala in K Tyr 268 Ala (TM6/TM7 loop; intracellular gate residue) mutation added to K (TS) Leu/Na b apo Leu/Na b apo shift (Fig 3b, Suppl. Fig 7a) n.d shift (Fig 3c, Suppl. Fig 7b) no shift (Suppl. Fig 7d) n.d shift (Suppl. Fig 2b) partial shift (Fig 3c, Suppl. Fig 7c) no shift (Suppl. Fig 2b) no shift (Fig 3d) n.d no shift no shift (Fig 3d) partial shift (Fig 3d, Suppl. Fig 8b) partial shift (Suppl. Fig 8c) partial shift (Suppl. Fig 8d) shift (Suppl. Fig 8a) 53.2 ± 3.7 nm 1.4 ±.1 µm d (ref 26) 41.5 ± 3.9 µm 53.3 ± 5.8 µm Yes Yes Yes No a Left shift of peak retention volume relative to uncomplexed on a gel filtration column indicating formation of complex. n.d, not determined. b 1 mm leucine, 2 mm NaCl c K d, dissociaiton constant, measured using scintillation proximity assay d K d determined by isothermal titration calorimetry e as judged by time course of leucine uptake in proteoliposomes with 5 mm NaCl gradient 1

2 doi:1.138/nature1737 Supplementary Table 2 Data Collection Statistics K (Y18F)- K (TSY)- K (TS) Beamline APS-24-ID-E APS-24-ID-E ALS 5..2 Space group P2(1)2(1)2(1) C222(1) C2 Cell dimensions 12.9, 162.8, 21.1 Å; 9, 9, , 169.8, 13.4 Å; 9, 9, , 87.3, 81.7 Å; 9, 95.7, 9 Wavelength (Å)* Resolution (Å)* ( ) ( ) 2.-3.( ) Completeness* 89.1(92.) 99.6 (1.) 98.5 (89.9) Multiplicity* 4.2(4.1) 3.8(3.8) 3.6(3.2) I/σI* 11.9(1.9) 17.8(2.) 14.3 (1.9) R merge *.16(.56).7(.69).84 (.517) Wilson B factor (Å 2 ) Refinement Statistics PDB code 3TT1 3TT3 3TU Refinement method Restrained, NCS, individual ADP, TLS Restrained, individual ADP, TLS Restrained, individual ADP Resolution (Å) Number of reflections R work /R free.219/ /.3.218/.288 Number of atoms (Light/heavy chains) 3312/ /1651 Detergent (SOG, OCT) b 64 8 Sum B factors (Å 2 ) (A/B) a 8.3/ Light chain (L/M) a 88.5/ Heavy chain (H/I) a 78.2/ r.m.s.d. values Bond lengths (Å) Bond angles ( ) Molprobity scores after adding hydrogens Ramachandran outliers (%)..11 c. Ramachandran favored (%) Clash score (percentile) Overall score (percentile) *Values in parentheses are for highest resolution shell. a Chains in the asymmetric unit b SOG: n-octyl- -D-thioglucoside; OCT: Octan-1-ol c Ser 7 of chain L of 2

3 doi:1.138/nature1737 Outward-open S Na+ Outward-occluded S Na+ inward-open Inward-occluded SI Figure 1: Alternating access mechanism of neurotransmitter-sodium symporters. Starting from the outward-open structure in the cycle, the transporter binds a substrate (S) and sodium ions, leading to the outward-occluded conformation. The next step in the transport cycle is the structural transition to the inward-occluded conformation, releasing sodium ions and substrate into the intracellular mileu. In the final step, the empty inward-open transporter re-orients to face the extracellular side to start a new transport cycle. 3

4 doi:1.138/nature1737 a b c S1 site Na2 site 3 T354 S355 Q361 8 Y18 1b 6a 1a 6b Trp fluorescence 2B12 K (Y18F) No K (TSY) Trp fluorescence 6A1 K (Y18F) No K (TSY) Intracellular gate Y268 SI Figure 2: mutants and conformation-specific antibody fragments. a, Location of the substitution mutations created for this study and their structural significance. b, c, Fluorescent-detection size exclusion chromatography (FSEC) analyses showing that the antibody fragments (s) used for crystallization are conformation-specific. Whereas both 2B12 and 6A1 s recognize in the absence of substrate and sodium, the 2B12 binds to K (Y18F) yet not to K (TSY) (b) and 6A1 recognizes K (TSY) but not K (Y18F) (c). 4

5 doi:1.138/nature1737 Out In 1 6b 1a 8 IL5 variable heavy chain variable light chain SI Figure 3: View of interface between the outward-open K (Y18F) and 2B12. Only the variable domains of the light and heavy chains of the are shown for simplicity. The 2B12 binds on the cytoplasmic face of, contacting residues on the N-terminus (residues 7-11), TM2 (residues 68, 71, 72), loop between TM6 and TM7 (residues 268, ), and IL5 of (residues 427, 43, 431, 434, 435, 438, 439, 441, 443) and burying 827 Å 2 of surface area in the interface. 5

6 doi:1.138/nature1737 Na2 Na1 SI Figure 4: Overlay of outward-open and Trp-bound structures showing that the two structures are similar. Helices in the outward-open, K (Y18F) structure are color-coded as per Figure 1, while the Trp-bound structure 12 (pdbcode 3F3A) is shown in light pink. Na ions at the Na1 and Na2 sites of the two structures are shown as purple (outward-open) or grey (Trp-bound) spheres. Note that the conformation of the intracellular loops in the two structures are very similar, indicating that the 2B12 (not shown here) bound to K (Y18F) does not substantially perturb backbone conformation. 6

7 doi:1.138/nature1737 a Specific [ 3 H] Leu binding cpm X K d = 41.5 ± 5.8 µm 15 1 K d = 53.3 ± 5.8 µm [Leu] µm b Trp fluorescence B12 K (Y18F) K + Leu, Na c K (TS) with 2B12 d 6A1 Trp fluorescence apo Leu, Na Trp fluorescence K K (TSY) K + Leu, Na K (TSY) + Leu, Na SI Figure 5: Mutations at Na2 site and analysis of conformation specific s. a, Saturation binding curves and nonlinear regression analysis for K (TS) (closed circles) and K (TSY) (open diamonds) obtained from radioligand binding experiments with [ 3 H]-L-leucine. c.p.m., counts per minute. Error bars, s.e.m.; n=3. b, FSEC traces of variants with or without substrate and sodium, and with 2B12 except the black trace of non-complexed K. c, FSEC profiles of K (TS) 2B12 complexes. The elution profile of non-complexed K (TS) is represented by the black trace. d, FSEC traces of variants with and without substrate and sodium, in the presence of the 6A1. 7

8 ffmol Leu/µg dfmol Leu/µg doi:1.138/nature1737 a b DPM 25, 2, 15, 1, 5, Background Total fmol Leu/µg c Time (min) Time (min) 4 Background 1 32 Total 8 DPM e DPM g Time (min) Background Total Time (min) Time (min) Time (min) -1. h DPM Background Total fmol Leu/µg Time (mins) Time (mins) SI Figure 6: [ 3 H]-L-leucine uptake in K (Y18F), K (TS), K (TSY), and K. Time course of leucine uptake measured in proteoliposomes with 5 mm NaCl gradient and either 2 nm ( K (Y18F)), 4 nm ( K (TS) and K (TSY)), or 1 nm [ 3 H]-L-leucine ( K ). In a, c, e, g, total and background disintegrations per minute (d.p.m) as a function of time for K (Y18F), K (TS), K (TSY), and K, respectively. In b, d, f, h, leucine uptake after background substraction for K (Y18F), K (TS), K (TSY), and K, respectively. The specific activity of [ 3 H]-L-leucine used in these experiments is the same. Error bars are s.e.m (n = 4-5). 8

9 doi:1.138/nature1737 Na2 Ala Na1 SI Figure 7: Alanine- and sodium-bound K (TS) adopts the outward-occluded conformation. Superposition of Ala- and Na + - bound structures of K (TS) (colored as in Figure 1) and wild-type (ref 11, pdbcode 3F48; grey colored). F o -F c omit density map is shown for Ala and Na + at the Na1 site in K (TS); map is contoured at 4σ. No omit density was detected in the Na2 site. Ala (wheat colored stick) and a Na + ion (purple sphere) were modeled into K (TS), coincident with the omit density. Na + ion at the Na2 site shown here as a grey sphere is from the 3F48 structure. 9

10 doi:1.138/nature1737 a b Trp Fluorescence K 1 mm Leu, 4 mm NaCl x.5x 1x 2x 5x 1x Trp Fluorescence K (TS) 1 mm Leu, 4 mm NaCl x.5x 1x 2x 5x 1x c d Trp Fluorescence apo K (TS) x.5x 1x 2x 5x Trp Fluorescence K (TSY) 1 mm Leu, 4 mm NaCl x.5x 1x 2x 5x SI Figure 8: Titration of variants with the 2B12 monitored by FSEC..27 µm was titrated with x,.5x, 1.x, 2.x, 5.x, and 1.x molar excess of. In b and c, the maximum shift of the 2B12 complex as typified by the K 2B12 complex is indicated by the grey dotted line. 1

11 doi:1.138/nature1737 a K (TSY) b K 2 2 Trp Fluorescence x.5x 1.x 2.x 5.x Trp Fluorescence x.5x 1.x 1.5x 2.x 5.x c d Trp Fluorescence K (Y18F) x.5x 1x 2x 5x Trp Fluorescence K (TS) x.5x 1x 2x 5x SI Figure 9: Titration of variants with the 6A1 monitored by FSEC in the absence of leucine and sodium..27 µm was titrated with 6A1 at x,.5x, 1x, 1.5x, 2x, or 5x molar excess over. In (a) K (TSY), (b) K, (c) K (Y18F), and (d) K (TS). In b, c, and d, the maximum shift undergone by the K (TSY) 6A1 complex is indicated by the grey dotted line. 11

12 doi:1.138/nature1737 C 12 N N 12 C SI_Figure 1: Crystallographic dimer in C222 1 crystals of K (TSY). While the asymmetric unit contains only one - complex, an antiparallel dimer, mediated largely by TM12 is formed by a crystallographic two-fold symmetry axis oriented parallel to the putative membrane plane. 12

13 doi:1.138/nature1737 EL4 Out 3 11 In IL b 1a variable light chain IL1 variable heavy chain SI Figure 11: K (TSY) 6A1 interface. The 6A1 binds to the intracellular face of, with its complementarity determining region inserted into the intracellular cavity of the inward-open state. The interface buries 12 Å 2 of surface area and involves residues from IL1 (68, 7-75, 83, 86), TM4/TM5 loop ( ), TM5 (191, 192), TM6 (264, 265, 267, ), TM8 (361, 364, 365, 368, 369), TM9 ( ), IL5 (427, 431, 434, 435, 438), and TM12 (58, 511). 13

14 doi:1.138/nature1737 a b F324 L29 F32 F324 W114 A319 R3 W114 L29 I111 Y18 L4 Y17 D44 D41 I111 Y18 F32 L4 A319 D41 Y17 SI Figure 12: S2 site is collapsed in the inward-open structure. a, View of the residues lining the putative S2 site in the inward-open structure. An unmodeled F o -F c difference density seen adjacent to the S2 site is shown here; map contoured at 3σ. b, Surface representation of the putative S2 site residues shows that there is no room for a leucine to be bound in this site. 14

15 doi:1.138/nature1737 TM1b TM1a SI Figure 13: Conformational change in TM1 upon transition from occluded to inward-open state in and Mhp1. TM1 of the outward-occluded state of Mhp1 and are shown in light blue and orange, respectively, while TM1 of the inward-open state of Mhp1 and are colored dark blue and red, respectively. Superposition of the structures using TM1b shows that TM1 moves as a rigid body in Mhp1 but not in. 15

16 doi:1.138/nature1737 SI Discussion Stabilization of inward-open conformation On the basis of the outward-open structure, we hypothesized that weakening of the Na2 binding site by mutation of Thr354 and Ser355 to valine and alanine, respectively, would shift the conformational equilibrium of towards the inward-open state. Functional characterization of K (TS) demonstrates that, while it exhibits greatly reduced substrate affinity (K d = 41.5 ± 3.9 µm, SI Figure 5a), it retains measurable substrate transport activity, like the K (Y18F) variant (SI Figure 6a-d). We note that because the K (Y18F) and K (TS) mutants bind substrate ~1-fold more weakly than K (refs.12, 25), we used 2 to 4-fold higher concentrations of substrate in the flux assays of these mutants. Indeed, even though the K (Y18F) and K (TS) mutants are active in flux measurements, they are likely severely compromised in both Km and Vmax in comparison to the wild type transporter. In accord with the capacity of the Na2 site mutant to bind and transport substrate, crystallographic analysis of the K (TS) complex with alanine shows formation of the canonical outward-occluded state (SI Figure 7). To probe how mutations at the Na2 site perturb the conformational equilibrium of, we carried out FSEC experiments using conformation-specific s. We first demonstrated that the 2B12 forms a stable complex with the outward-occluded state of the wild-type-like K variant and that this complex co-migrates with the apo K (Y18F) species (SI Figure 5b). We next asked whether the 2B12 binds to the Na2 mutant, K (TS), finding that while it forms a stable complex in the presence of leucine and sodium, complex formation is diminished in the absence of substrate and sodium, as shown by a broadening of the 16

17 doi:1.138/nature1737 peak (SI Figure 5c, SI Figure 8). Taken together, these data suggest that the conformational equilibrium of the K (TS) mutant is shifted away from an outward-open conformation. Nevertheless, partial binding of the 2B12 to the apo K (TS) mutant implies that the intracellular gate can also occupy a closed conformation, like that visualized in the K (Y18F) 2B12 crystal structure. Therefore, we further weakened this gate by mutation of Tyr268 to Ala 27, yielding the K (TSY) variant. The K d of K (TSY) for leucine is 53.3 ± 3.8 µm (SI Figure 5a), similar to that of K (TS), yet it is unable to drive sodium-coupled substrate transport (SI Figure 6e, f). The retention of substrate binding activity supports the notion that K (TSY) is folded and that it occupies a native-like conformation. Most importantly, K (TSY) binds to the 6A1 in the absence of leucine and Na +, but not in the presence of 1 mm leucine and 2 mm NaCl (SI Figure 5d). Furthermore, 6A1 binds to K (TS), K, and K (Y18F) albeit with diminishing affinity, in the absence of leucine and sodium yet not with substrate and sodium ions (SI Figure 5d, SI Figure 9). These results suggest that the 6A1 selectively binds to a conformational state of the K (TSY) mutant that is also populated by the wild-type transporter. 17

18 doi:1.138/nature1737 Supplementary Movie Legend The movie depicts the conformational changes associated with isomerization from the outwardopen, substrate-free state to the outward-occluded state and then to the inward-open state. The movie was made by morph transitions from the substrate-free K (Y18F) outward-open structure to the leucine-bound, outward-occluded state structure (PDB code 2A65) and then to the inward-open K (TSY) structure using LSQMAN of the Uppsala Software Factory ( The transport cycle beings in the substrate-free, outward-open state in which sodium ions are bound at the Na1 and Na2 sites, the extracellular gate is open with Phe 253 flipped away from the binding pocket and the intracellular gate is closed. The cycle ends in the inward-open state from which substrate and sodium ions are released into the cytoplasm. The conformational transitions that undergoes to capture leucine and sodium ions from the extracellular space and release them into the cytoplasm are shown in four different views of the transporter: the first two views are in the plane of the membrane, the third looking down into the binding pocket from the extracellular side and the fourth is looking up into the binding pocket from the intracellular side. is colored with the same scheme used in Figure 1. The sodium ions are shown as purple spheres while the substrate leucine is depicted in CPK representation. The extracellular and intracellular gating residues are shown in stick representation. 18