Figures and data in Allosteric effects of the coupling cation in melibiose transporter MelB

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Tables
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9 figures, 1 table and 7 additional files

Figures

Figure 1

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Functional characterizations.

(a) α-NPG downhill transport. *E. coli* DW2 cells with the induced α-galactosidase by melibiose in the absence or presence of WT MelB_St 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 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 the supernatant released from the cells was measured at A₄₀₅ nm. The mean values from two tests were plotted against incubation time with standard error bars. (b) [³H]Raffinose active transport. The *E. coli* DW2 cells in the absence or presence of MelB_St 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. The mean values from two tests were plotted against the incubation time with standard errors. (**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 MelB_St or D59C mutant was placed in the reaction cell, and methyl α-D-galactoside (α-MG) or raffinose at 10 mM (against the WT, left column) or 100 mM (against D59C, right column) from the syringe was 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 MelB_St (red for α-MG and blue for raffinose) or to buffer (light blue). The heat change ∆Q (µJ; filled black symbol) was plotted against the mole ratio of the sugar to MelB_St (top/right axes in green). The K_d values were the average of two tests with standard error. (c) α-MG. (d) Raffinose. Source data are available for panels a-d as Figure 1—source data 1a–d.

Figure 1—source data 1 Excel tables for transport and ITC curves.: https://cdn.elifesciences.org/articles/108335/elife-108335-fig1-data1-v1.xlsx
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Figure 2 with 1 supplement

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

***Upper row***: Cartoon representation of the structures of D59C MelB_St bound with α-NPG, melibiose (α-disaccharide), and raffinose (α-trisaccharide), respectively, along with a surface presentation of D59C MelB_St 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.

Figure 2—figure supplement 1

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Overlay.

Four crystal structures of the D59C MelB_St with melibiose, α-MG, raffinose, and α-NPG bound, respectively, were superimposed with the MRSD values <0.4 Å. Top, side view with a rainbow color code from N- to C-termini. Bottom, viewed from the cytoplasmic side.

Figure 3 with 1 supplement

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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) was contoured at a level of σ=1.2. Dashed lines indicate distances within hydrogen-bonding or salt-bridge interactions (Å).

Figure 3—figure supplement 1

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Waters interacting with the salt-bridge network.

Nine charged residues, including the three N-terminal residues (Lys138, Arg141, and Glu142 on helix V) colored in yellow and the six C-terminal residues (Arg295, Asp351, Asp354, Glu357, Rrg363, and Glu365 on helix XI and Loop_10-11) colored in cyan, are presented in sticks and highlighted in surface representation in light blue. Red sphere, water molecule. Wat 1 is bound with α-NPG and Thr373; Wat 2 and Wat 3 are associated with the salt-bridge pair Arg295 and Asp351, respectively; and Wat 4 is interacting with Lye138. The α-NPG is colored yellow and labeled. The transmembrane helices are labeled in roman numerals. (a) Side view; (b) Top view.

Figure 4 with 1 supplement

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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.

Figure 4—figure supplement 1

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Overlay of the bound melibiose and α-NPG.

The crystal structures of D59C MelB_St with melibiose or α-NPG bound were aligned, and the bound substrates were highlighted. The Tyr26 on helix I stacks with the phenyl ring of α-NPG.

Figure 5 with 1 supplement

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Deuterium uptake of the apo MelB_St.

HDX experiments on WT MelB_St were conducted as described in Methods. All values were from the mean of six measurements at the apo state. (a) Deuteration map of the apo MelB_St. Mean deuteration levels of MelB_St peptides at the apo state averaged first across timepoints (30 s, 300 s, 3000 s), then across two replicates, were presented against amino-acid residue sequence. The values at both N- and C-terminal regions greater than 1.2 are shown 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. Source data are available as Figure 5—source data 1 and 2.

Figure 5—source data 1 HDX_MS raw data_apo & Na.: https://cdn.elifesciences.org/articles/108335/elife-108335-fig5-data1-v1.xlsx
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Figure 5—source data 2 HDX-MS raw data - apo, mel, mel&Na.: https://cdn.elifesciences.org/articles/108335/elife-108335-fig5-data2-v1.xlsx
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Figure 5—figure supplement 1

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Mapping of HDX data on an inward-facing melibiose-bound D59C MelB_St.

(a) Non-covered residues. Transmembrane helices are labeled in Roman numerals. The non-covered positions on the N-terminal domains and C-terminal domain, including extended loops, are shown in α-carbon position and colored in blue and pale cyan, respectively. (b) Overlapping peptide with ligand-induced protection and deprotection of deuterium uptakes. Peptides with ΔD value greater than the absolute value of the threshold and p<0.05 at any time point were colored according to the legend on the figure. The membrane region of MelB_St is indicated by a gray bar. Each peptide or overlapping peptide was indicated by arrows pointing to the starting position of the peptide.

Figure 6 with 3 supplements

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Residual plots and structural mapping.

HDX experiments on WT MelB_St in the apo or holo states (with melibiose, Na⁺, or Na⁺ plus melibiose) were conducted as described in Methods. (a) Residual plots (D_{Holo - 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 s, respectively; dark gray curve, total uptake from all three time points. Deprotection, ΔD_{Holo – Apo} > 0; protection, ΔD_{Holo – 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 D_{Mel – Apo}, D_{Na(+) – Apo}, and D_{Na(+)Mel – Apo}, respectively. The red star symbol indicated the location of the Na⁺-binding pocket, the drawing lines represented the disordered MelB_St C-terminal tail, and a gray bar showed the membrane region of MelB_St. 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 s and red for data from 30 s). Non-covered residues from each dataset are shown as gray spheres at the Cα position and listed in Supplementary file 3, 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.

Figure 6—figure supplement 1

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Deuterium uptake time course of all peptides ΔD value >threshold.

The percentage of deuterium uptake measured in the absence (filled black square) or presence of melibiose (filled green circle), Na⁺ (filled red circle), or melibiose and Na⁺ (filled magenta circle), was plotted against labeling times of 0, 30, 300, and 3000 s. The peptide sequences and position were shown. (a) Melibiose vs. apo. (b) Na⁺ vs. apo. (c) Melibiose and Na⁺ vs. apo. The y-axis scale was not identical.

Figure 6—figure supplement 2

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Figure 6—figure supplement 3

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Figure 7

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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 and Na⁺ (filled blue square) was plotted against labeling times of 0, 30, 300, and 3000 s. 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. black star, 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.

Figure 8 with 1 supplement

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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 and Na⁺ (filled blue square) was plotted against labeling times of 0, 30, 300, and 3000 s. 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 (Loop_4-5/IV), Arg295 (IX), Asp351, Asp354, and Glu357 (X), and Arg363 and Asp365 (Loop_10-12) 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. Model at the left side, helices V and VIII in front; model at the right side, helices II and XI in front. (c) HDX and ligand effects mapping on an outward-facing topology model of MelB_St. Melibiose- and Na⁺-binding residues were labeled in red or black, respectively, and residues in the gating salt-bridge network were labeled in gray. Residues with higher levels of deuteration and ligand-induced protections were highlighted in yellow background, and residues with low levels of deuteration and no ligand effects were colored in cyan background. This topological figure was modified from the Figure 3—figure supplement 4 of the article ‘Mobile barrier mechanisms for Na⁺-coupled symport in an MFS sugar transporter’ published by eLife (Hariharan et al., 2024b).

Figure 8—figure supplement 1

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The gating and sugar-binding residue Arg149.

Arg149 was shown at the aligned outward- and inward-facing conformations. Arg149 side chains in red or blue were from the aligned inward-facing [PDB 8T60] and the melibiose-bound outward-facing conformations, respectively.

Figure 9

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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 a 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 was performed and the p-values were presented.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Escherichia coli)	DW2	Botfield and Wilson, 1988	melA⁺ melB^- lacZ^-Y^-
Recombinant DNA reagent	pK95/ΔAH/WT MelB_St/CHis₁₀	Guan et al., 2011		Protein expression
Recombinant DNA reagent	pK95/ΔAH/D59CMelB_St/CHis₁₀	Ethayathulla et al., 2014		Protein expression
Chemical compound, drug	Melibiose	Acros Organics (Thermo Fisher Scientific)	Cat# 125375000	Crystallization
Chemical compound, drug	[³H]Melibiose	Perkin-Elmer	Radiolabeled
Chemical compound, drug	[³H]Raffinose	American Radiolabeled Chemicals (ARC)	Radiolabeled, Cat# ART 0229
Chemical compound, drug	Raffinose	Research Products International Corp., (RPI)	Cat# R20500	Crystallization
Chemical compound, drug	α-Methyl galactoside (α-MG)	Sigma-Aldrich	Cat# M1379	Crystallization
Chemical compound, drug	p-Nitrophenyl α-D-galactoside (α-NPG)	Sigma-Aldrich	Cat# N0877	Crystallization
Chemical compound, drug	Undecyl-β-D-maltopyranoside (UDM)	Anatrace	Cat# U300
Chemical compound, drug	Dodecyl-β-D-maltopyranoside (DDM)	Anatrace	Cat# D310
Chemical compound, drug	E. coli lipids	Avanti Polar Lipids, Inc	Extract Polar, Cat# 100600	Crystallization
Chemical compound, drug	Polyethylene glycol 400 (PEG400)	Hampton Research	Cat# HR2-603	Crystallization
Commercial assay or kit	Micro BCA Protein Assay	Pierce Biotechnology, Inc
Software, algorithm	ccp4i2 program	Potterton et al., 2018	X-ray data reduction and analysis
Software, algorithm	Phenix (1.21)	Liebschner et al., 2019	Molecular replacement and structure refinement
Software, algorithm	Coot (0.9.8.96)_	César-Razquin et al., 2015	Model building
Software, algorithm	BioPharma Finder software (v 5.1)	Thermo Fisher Scientific	MS data process
Software, algorithm	HDExaminer	Sierra Analytics	HDX-MS data process
Software, algorithm	MATLAB (In-house script)	Thermo Fisher Scientific	HDX-MS data process
Software, algorithm	Pymol (3.1.5.1)	Schrodinger, 2013	Molecular visualization program
Software, algorithm	UCSF ChimeraX (1.10)	Pettersen et al., 2021	Molecular visualization program
Software, algorithm	Origin 2024		Graphical software
Software, algorithm	AMBER24 software package	Case et al., 2023	MD simulation