Functional characterizations.

(a) α-NPG down-hill transport. E. coli DW2 cells with the induced α-galactosidase by melibiose in the absence or presence of WT MelBSt or D59C mutant were washed before incubating with 0.5 mM p-nitrophenyl-α-D-galactoside at 30 °C in the absence or presence of 20 mM NaCl or 20 mM LiCl as described in Methods. The cell-aliquots at given time points at 0, 1, 5, 10, 20, 30, and 60 min were quenched with 0.3 M Na2CO3, followed by centrifugation to remove the cells. The p-nitrophenol in supernatant released from the cells was measured at A405 nm. (b) [3H]Raffinose active transport. The E. coli DW2 cells in the absence or presence of MelBSt with no α-galactosidase induction were used for the active transport of [3H]raffinose at 1 mM (specific activity, 10 mCi/mmol) at 23 °C in the absence or presence of 50 mM NaCl or LiCl. The cellular uptake time course measurements at 0, 0.08, 0.17, 0.5, 1, 2, 5, 10, and 30 min were carried out by a dilution and fast-filtration method. (c & d) ITC measurement of α-MG (c) or raffinose (d). ITC measurements were performed at 25 °C under similar buffer conditions: 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, and 0.035% UDM detergent. For each experiment, 80 µM of the purified WT MelBSt or D59C mutant was placed in the reaction cell, and methyl α-D-galactoside (α-MG) or raffinose at 10 mM (against the WT) or 100 mM (against D59C) from the syringe are incrementally titrated to generate the thermograms. The curve fitting is performed with a one-site independent-binding model included in the NanoAnalyze software (version 3.7.5). The thermograms were plotted as baseline-corrected heat rate (µJ/sec; left axis) vs. time (bottom axis) for the titrant to MelBSt (red for α-MG and blue for raffinose) or to buffer (light blue). The heat change ΔQ (µJ; filled black symbol) was plotted against the mole ratio of the sugar to WT MelBSt (top/right axes in green). (c) α-MG. (d) Raffinose.

Crystal structures of D59C MelBSt in complex with α-sugars substrates.

Upper row: Cartoon representation of the structures of D59C MelBSt bound with α-NPG, melibiose (α-disaccharide), and raffinose (α-trisaccharide), respectively, along with a surface presentation of D59C MelBSt complexed with α-methyl galactoside (α-MG). All structures were oriented with the cytoplasmic side on top and the N-terminal domain (colored green) on the left. Each sugar molecule is colored yellow. The blue sticks and surfaces indicate residues within 5 Å of the sugar molecules. Lower row: Sugar-binding pocket. Residues from the N-terminal and C-terminal domains were shown in surface representation, and colored in green and blue, respectively.

α-NPG binding.

The cross-eye stereo view of α-NPG binding. The side chains forming the binding pocket formed from N- and C-terminal domains were shown in stick representation, colored according to corresponding hosting helices in rainbow, and labeled in black and blue, respectively. Isomesh map of the α-NPG (in yellow) and Wat 1 (in red) were contoured at a level of σ =1.2. Dashed lines indicate distances within hydrogen-bonding or salt-bridge interactions (Å).

Binding of melibiose, α-MG, and raffinose.

All residues within a 5-Å distance to the bound substrates were shown in sticks. Dashed lines, the distances within hydrogen-bonding and salt-bridge interactions (Å). (a) Melibiose binding. (b) α-MG binding. (c) Raffinose binding. (d) Residues in the sugar-binding pockets from the alignment of all four structures. (e) Substrates from the alignment of all four structures. Carbon positions on the galactosyl (C1-6) and glucosyl moiety (C1′-6′) were labeled on the melibiose molecule. Trp342 was removed for clarity in panels a, b, and d. Isomesh maps for each sugar and Asp19 were contoured at levels of σ = 1.5 for melibiose and raffinose or σ = 1.0 for α-MG.

Deuterium uptake of the apo MelBSt.

HDX experiments on WT MelBSt were conducted as described in Methods. All values were from the mean of six measurements at the apo state. (a) Deuteriation map of the apo MelBSt. Mean deuteration levels of MelBSt peptides at the apo state averaged first across timepoints (30s, 300s, 3000s), then across two replicates, were presented against amino-acid residue sequence. The values at both N- and C-terminal regions were greater than 1.2 showed as black bars. The chemical properties of the peptide-covered residue were indicated by background shading; the white background indicated the non-covered positions. (b) Relative deuterium uptake per peptide plot. The corresponding transmembrane helices and a few loops were marked. Notably, due to the nature of overlapping peptides, the indicated amino-acid position is not sequential. Peptides with greater deuterium uptake were labeled. Peptides covering sugar- and cation-binding sites were colored in green and blue, respectively, and the dynamic regions with greater uptakes were colored in red.

Residual plots and structural mapping.

HDX experiments on WT MelBSt in the apo or holo states (with melibiose, Na+, or Na+ plus melibiose) were conducted as described in Methods. (a) Residual plots (DHolo - Apo). Differential deuterium uptakes (ΔD) of each time point and the total uptake calculated from paired conditions for position 2-470 were plotted against the peptide number. Black, cyan, and purple bars, the deuterium uptake at 30, 300, and 3000 sec, respectively; dark gray curve, total uptake from all three time points. Deprotection, ΔDHolo – Apo > 0; protection, ΔDHolo – Apo < 0. Each sample was analyzed in triplicate. Dashed lines indicate the levels of the global threshold values calculated from each dataset, as labeled. The protein residue positions corresponding to the overlapping peptides were marked, and the covered transmembrane helices were labeled in Roman numerals. All deprotected peptides with statistical insignificance (ΔD >threshold and P<0.05) in each dataset were labeled. (b-d) Peptide mapping on the crystal structure of the melibiose-bound inward-facing conformation for DMel – Apo, DNa(+) – Apo, and DNa(+)Mel – Apo, respectively. The red star symbol indicated the location of the Na+-binding pocket, the drawing lines represented the disordered MelBSt C-terminal tail, and a gray bar showed the membrane region of MelBSt. The peptides of ΔD values with statistical significance (ΔD > |threshold| and P < 0.05) at any timepoint are highlighted either in ribbon representation for protection (colored in blue for peptides covering sugar-binding pocket and in green for all other regions) or in cartoon representation for deprotection (colored pink for data from 3000 sec and red for data from 30 sec). Non-covered residues from each dataset are shown as gray spheres at the Cα position and listed in Table S3, and peptides with statistically insignificant differences (either ΔD < |threshold| or P>0.05) are illustrated in backbone representation in gray. Peptide positions are marked by their starting residue; the number of overlapping peptides is indicated in round brackets, and the location is shown in square brackets.

Allosteric effects on substrate-binding sites.

Deuterium uptake time course of representative peptides covering the sugar- and cation-binding pockets. The percentage of deuterium uptake measured in the absence (empty black square) or presence of melibiose (Mel), Na+, or Mel & Na+ (filled blue square) was plotted against labeling times of 0, 30, 300, and 3000 sec. The peptide sequences were shown with residues at the cation- or sugar-binding pocket highlighted in black or red, respectively. Lys377 between the two binding pockets was highlighted in gray. P values are provided for each time point where the ΔD value exceeds the threshold. On the melibiose-bound structure, residues participating in the sugar binding and cation binding were shown in stick. Red start, the cation-binding pocket; red text labels: residues showing greater HDX with significant substrate effects; black text labels: residues showing poor HDX with no significant substrate effects.

Allosteric effects on gating salt-bridge network.

(a) Deuterium uptake time course of representative peptides covering the cytoplasmic gating salt-bridge network. The percentage of deuterium uptake measured in the absence (empty black square) or presence of melibiose (Mel), Na+, or Mel & Na+ (filled blue square) was plotted against labeling times of 0, 30, 300, and 3000 sec. P values are provided for each time point where the ΔD value exceeds the threshold. The peptide sequences were shown with residues at the salt-bridge network or sugar-binding pocket highlighted in purple or red, respectively. On the α-NPG bound structure, the gating salt-bridge network (Lys138, Arg141, and Glu142 (Loop4-5/IV), Arg295 (IX), Asp351, Asp354, and Glu357 (X), and Arg363 and Asp365 (Loop10-12) and their polar contacts with the backbone of other positions were shown in dashed lines. The N- and C-terminal domains were colored in light cyan and gray, respectively. The residues at the cation binding pocket were shown in ball and stick, as also indicated by the red star. Wat, water. Three sugar-binding residues, Asp19, Arg149, and Gln272, were shown in stick. Arg363 is in the list of uncovered positions. The region covering both the cytoplasmic gating salt-bridge network and sugar-binding residue Arg149 was highlighted in solid color, and the peptide 284-291 at helix III-ICH2 was also highlighted in pink. (b) Deprotection at loops. Deuterium uptake time course of four peptides at loops, mainly at the gating area, was presented and also mapped on the outward-facing structure, which was overlayed with the inward-facing structure [PDB 8T60]. The cytoplasmic and periplasmic gates were indicated. The peptides with deprotection by substrate were colored in pink. (c) HDX and ligand effects mapping on an outward-facing topology model of MelBSt. Melibiose-and Na+-binding residues were labeled in red or black, respectively. Residues showing ligand-induced protection and deprotection were colored in blue or red, respectively.

Side-chain flexibility analyzed from MD trajectories.

For each residue, the side-chain RMSF values in each of the five replicas for the apo and melibiose- and Na+-bound states were plotted. The mean of the RMSF values in each state is represented as red circle (apo state) or blue square (melibiose- and Na+-bound state). For each residue, unpaired t test of the RMSF mean values between the apo and the melibiose- and Na+-bound states were performed and the p-values were presented.