The sugar analog α-NPG has been determined as a substrate for MelB_Ec by measuring p-nitrophenol production from the cells that expressed both MelB_Ec and α-galactosidase (Wilson and Wilson, 1987). The same assay was modified to determine the α-NPG translocation mediated by MelB_St in DW2 cells. Melibiose at 1 mM was added to induce α-galactosidase expression during cell growth, and the washed cells were applied to measure the time course of p-nitrophenol release into the media upon adding 1 mM α-NPG into the induced cells. The detection was for the p-nitrophenol, which resulted from α-NPG transport and hydrolysis. The results showed that both WT MelB_St and D59C uniport mutant mediated the translocation of α-NPG (Figure 1a; Figure 1—source data 1a) at this downhill mode of transport.

To determine raffinose transport activity, [³H]raffinose transport in the E. coli DW2 strain was carried out in the absence of Na⁺ and Li⁺ or the presence of Na⁺ or Li⁺ (Figure 1b; Figure 1—source data 1b). The results showed that raffinose, a trisaccharide formed from melibiose and fructose, is also a substrate and transported by MelB_St.

Previously, our ITC studies in the presence of Na⁺ revealed that the binding affinity, K_d values, of the WT and D59C MelB_St for melibiose were 1.25 mM ± 0.05 mM and 4.96 ± 0.11 mM, respectively, and for α-NPG were 16.46 ± 0.21 µM or 11.97 ± 0.09 µM, respectively (Hariharan and Guan, 2017; Guan and Hariharan, 2021; Hariharan and Guan, 2014; Hariharan and Guan, 2021). The same assay was utilized to determine the α-MG and raffinose binding in the presence of Na⁺ (Figure 1c–d; Figure 1—source data 1c-d). By injecting the α-MG or raffinose in a buffer-matched solution into the sample cell containing the WT MelB_St or the D59C uniporter mutant in the presence of Na⁺, the isotherm curve fitted well, yielding K_d values of 0.88 ± 0.04 mM or 3.79 ± 0.53 mM, for α-MG or raffinose, respectively. Raffinose, which has more sugar units, exhibits a poor binding affinity to MelB_St.

The uniporter D59C MelB_St mutant exhibits greater thermostability, making it a valuable tool for structural analysis of sugar binding (Guan and Hariharan, 2021). Here we report four crystal structures of D59C MelB_St with the endogenous sugar substrate melibiose, and two other α-galactosides, methyl α-galactoside (α-MG) with a single sugar unit or raffinose with three sugar units, as well as α-NPG at an improved resolution. The structure statistics were presented in Supplementary file 1. All structures adopt a virtually identical outward-facing conformation with RMSD values of less than 0.4 (Figure 2—figure supplement 1). This typical MFS-fold transporter with 12 transmembrane helices is organized into two six-helix domains linked by the middle loop between helices VI and VII (Loop_6-7). There are three cytoplasmic helices at the loop_6-7, loop_8-9, and the C-terminal tail or lid (Figure 2), named ICH1-3, respectively; and ICH1 and ICH2 run parallel to the membrane bilayer. In all structures, one sugar molecule is bound in the middle of the protein and sandwiched by both N- and C-terminal domains as described previously (Guan and Hariharan, 2021). As indicated by the sliced surface, the binding residues located within the cytoplasmic leaflet of both domains define the edge of the inner barrier, which prevents the sugar from passing across the transporter into the cytoplasm. On the periplasmic side, the open vestibule connects the solvent to the binding pocket. In the α-NPG-bound structure, as reported in PDB ID 8FRH (D59C apo structure) or ID 8FQ9 D55C mutant with DDMB (Hariharan et al., 2024a), a PEG molecule was modeled to the density near Arg296 along with helix IX, which could indicate a potential lipid-binding site (Figure 2—figure supplement 1).

To determine the structural dynamics of MelB_St and the influence of melibiose and/or Na⁺, the differential HDX-MS experiments were employed between MelB_St alone and MelB_St with melibiose and/or Na⁺ (Masson et al., 2019; Jia et al., 2020; Zmyslowski et al., 2022; Supplementary file 2). MelB_St residues 2–470 out of 476 were covered, achieving a coverage of over 87.47% and 86.62% from 149 or 152 overlapping deuterium-labeled peptides (Supplementary files 2 and 3; Figure 5—figure supplement 1, Figure 5—source data 1 and 2). There are 59, 63, or 59 non-covered residues for the datasets of the apo vs. melibiose-bound, Na⁺-bound, and melibiose- and Na⁺-bound states, respectively. Most are at the transmembrane helices IV, IX, X, and XI, and some cytoplasmic loops. Labeled peptides cover all periplasmic loops.

Three systems, including the apo, melibiose-bound, and melibiose- and Na⁺-bound states of a generated WT MelB_St, were constructed by embedding in a POPE:POPG (7:2) lipid bilayer. For each system setup, five independent replicas of MD simulations were carried out, each for ~400 ns, and the total sampling time for each system was ~2 μs.

Dehydration of sugar molecules is expected for binding; however, full or partial dehydration to bind MelB is unknown. The 2.60 Å resolution α-NPG-bound structure showed a partially dehydrated sugar at the binding site. The bound water connected the OH-4 and OH-6 on the galactopyranosyl ring with Thr373 and Gln372 at helix XI, and it was also surrounded by Asp124 at helix IV. Previous Cys-scanning mutagenesis has shown that the T373C mutant retained most activities. Still, a Cys residue on the Gln372 position significantly decreased the transport initial rate, accumulation, and melibiose fermentation, with little effect on protein expression (Markham et al., 2021), which supported the role of Wat-1 in binding. Notably, the orientation of OH-4 is crucial for distinguishing between galactose and glucose. Previously, the OH-4 has been shown at hydrogen-bonding distances from the carboxyl group of Asp124 and the indole group of Trp128 at helix IV (Guan and Hariharan, 2021; Hariharan et al., 2024a), and the identification of Wat-1 in this study added the polar residues Gln372 and Thr373 on helix XI to stabilize the critical OH-4 (Figure 3). Thus, these interactions define the specificity of galactosyl molecules, and Wat-1 is likely part of sugar binding. Notably, the OH-4 and OH-6 and Wat 1 are in close proximity to the Na⁺-binding residue Thr121 and the cation-binding important residue Lys377, implying that the Wat-1 is between the two specificity-determining pockets. As shown previously, the OH-3 and OH-2 at the opposite edge of the galactopyranosyl ring form multiple hydrogen-bonding interactions with the charged residues Asp19 and Arg149 at helices I and V, which can be assigned to play a crucial role in stabilizing the recognition of OH-4 and enhancing the binding affinity.

HDX-MS is a powerful technique that simultaneously discloses the dynamic information in various areas of a protein. The qualitative method overcomes the drawbacks of most site-specific labeling-based techniques; however, it only provides the conformational dynamic information on the equilibrium of all ensembles. In the current study, we determined the deuterium uptake rates of the full-length MelB_St in the absence or presence of melibiose and/or Na⁺, identified major dynamic regions (Figure 5), as well as substrate-induced effects on the substrate-binding sites and structural elements critical for conformational transition, including the cytoplasmic salt-bridge network and the gating areas (Figures 6—8).

Our studies of HDX complemented with molecular dynamics simulations on side-chain fluctuations showed that the Na⁺-binding pocket and the sugar-binding residues near the Na⁺-binding site at helix IV are conformationally rigid since they exhibited low deuteration level for both unbound and bound states (Figures 5—7 and 9; Supplementary files 3 and 4), which are consistent with the previous HDX-MS results of conformation transition (Hariharan et al., 2024b). Those rigidities are likely derived from their hosting helices (II and IV). In addition, the Na⁺-binding affinity, different from sugar binding, exhibited little difference between the inward- and outward-facing states (Hariharan et al., 2024b). The conformational freedom of the cation-binding site and most sugar recognition positions were likely restricted in most states to facilitate sugar binding.

The sugar-binding residues, located away from the Na⁺-binding site, are dynamic, exhibiting higher deuterium levels and side-chain fluctuations. Most of those positions are within the six dynamic regions identified from the apo state, including Lys18, Asp19, Ile22, Tyr26, Arg149, Ala152, Gln272, and Thr373 at helices I, V, and XI. Those regions were protected to varying extents by melibiose or Na⁺, and the protection was significantly greater when bound to both substrates (Figures 5—7). Even their conformational flexibility was restrained by melibiose and Na⁺, but the deuterium uptakes at those regions were still greater than those of peptides covering the cation-binding site (Figure 7). Notably, substrate-induced protection was also detected in other areas of the hosting helices I and V, as further discussed below.

Abundant peptides covering Arg149 consistently showed protection by melibiose or Na⁺, especially when both were present (Figures 5—8). Structurally, Arg149 is near to a stretch of charged residues as shown by this peptide 137-DK¹³⁸RER¹⁴¹E¹⁴²QLVPFPRF-150 (Figures 5—7), where the Lys138, Arg141, and Glu142 have been determined to play critical roles in the functionally crucial cytoplasmic salt-bridge network (Figure 8). The dynamics of Arg149 are likely modulated by this charged network and belong to the same dynamics group, which provided the structural basis for coupling between the sugar-binding affinity and protein dynamics. At the inward-open structure, this network is deformed. Arg149, as part of this gate, exhibits a large displacement due to an otherwise steric collision with helix X at the outward-facing state (Figure 8—figure supplement 1). The sugar-binding affinity at this inward-facing state was reduced by ~30-fold due to the broken binding pocket and the displacement of Arg149 and Gln372 (Hariharan et al., 2024b). Functionally, the single-site R149C mutant inhibited but did not eliminate the melibiose transport (Ethayathulla et al., 2014), and single-Cys149 (Markham et al., 2021) also fermented melibiose with intact cells. The neighboring P148L mutant decreased transport V_max with a better K_m value (Jakkula and Guan, 2012). All data showed that the dynamics of this charged network and sugar-binding affinity are interconnected.

This cytoplasmic gating salt-bridge network has a rigid center dictated by helix XI and dynamic surroundings from helices V, IX, XI, and loops 4–5, 8–9, 6–7, 10–11, and those dynamic positions were significantly inhibited by melibiose, Na⁺, especially the binding of both. Notably, Nb725 binding to the inward-facing conformation also inhibited the dynamics of the similar regions, as shown by peptides 137–150, 284–289, and 364–368 (Hariharan et al., 2024b). The similar HDX profiles from all testing conditions suggested the protein motions followed a similar pathway of the conformational transition. Thus, the dynamics of Arg149 are linked to motion and conformational transitions, as well as sugar-binding affinity. Connectively, it is postulated that Na⁺ binding between helices II and IV stabilizes the dynamics of the gating salt-bridge network and the flexibility of Arg149 and Asp19, which are located more than 10 Å away, thereby allosterically switching the primary substrate-binding site to the higher-affinity state.

The binding of sugar further stabilizes this network and promotes Na⁺ binding by preventing Na⁺ from leaving the cation-binding pocket, thus increasing Na⁺-binding affinity (Ethayathulla et al., 2014; Liang and Guan, 2024). Therefore, the cooperative binding of sugar and Na⁺ is part of the substrate-induced conformational allostery, and all functions together.

There are a few regions with substrate-induced deprotections. Two gating areas were deprotected by melibiose and Na⁺, including the cytoplasmic ICH2 and Loop_1-2, which suggested the structural arrangements. Notably, ICH2 is linked with Arg295 at one end of the salt-bridge network, and the dynamics of this area could influence the stability of the gating network, which might trigger the separation and conformational transition to the inward-open state. Thus, the increased dynamics of loop_1-2 and Loop_8-9 at both gating areas by melibiose, especially with Na⁺, can be interpreted as the tendency to form a transition-competent conformation with closing at the periplasmic gate and opening at the cytoplasmic gate. A region at the loop_6-7 responding to the ligand binding identified by the atomic force microscopy was not observed in this HDX study (Blaimschein et al., 2023).

The symporter H⁺-coupled lactose permease LacY and xylose permease XylE also show protonation dependence of sugar-binding affinity (Jia et al., 2020; Smirnova et al., 2008; Grytsyk et al., 2017), with separate cation- and sugar-binding sites (Guan and Kaback, 2006; Guan et al., 2007; Mirza et al., 2006; Kumar et al., 2014; Sun et al., 2012). Both transporters favored inward-facing conformation, and substrate binding induced outward-facing conformation, as indicated by the extensive HDX analysis with XylE (Jia et al., 2020; Jia et al., 2023) and a bunch of biophysical measurements of LacY (Smirnova et al., 2011). Notably, several dynamic regions in MelB_St were also identified in XylE; both proteins showed binding-induced structural changes in their loop_1-2 and loop_8-9 (Jia et al., 2023). In MelB_St, while no quantified information on the ensembles, all experimental results and molecular simulations on the minimum free-energy landscape for sugar translocation suggested that the apo MelB favors the outward-facing conformation, and the outward-facing conformation is further preferred when bound with melibiose and Na⁺ (Guan and Hariharan, 2021; Hariharan et al., 2024b; Liang and Guan, 2024).

Our studies on structure, HDX, and MD simulations allow us to conclude that the sugar-binding affinity of MelB_St is coordinated with protein structural motions and conformational transition between inward- and outward-facing states. Na⁺ binding restrains the dynamics of remote sugar-binding residues via stabilizing the dynamic cytoplasmic salt-bridge network, thereby increasing sugar-binding affinity allosterically. The conclusion provides insightful knowledge to understand cooperative binding and symport mechanisms.

Melibiose was purchased from Acros Organics (Thermo Fisher Scientific). [³H]Raffinose and label-free raffinose were purchased from American Radiolabeled Chemicals (ARC), Inc and Research Products International (RPI) Corp., respectively. Raffinose (RPI Chemicals), α-methyl galactoside (α-MG), and p-Nitrophenyl α-D-galactoside (α-NPG) were purchased from Sigma-Aldrich. Detergents undecyl-β-D-maltopyranoside (UDM) and dodecyl-β-D-maltopyranoside (DDM) were purchased from Anatrace. E. coli lipids (Extract Polar, 100600) were purchased from Avanti Polar Lipids, Inc All other materials were reagent grade and obtained from commercial sources.

E. coli DW2 cells (melA⁺B^-, lacZ^-Y^-) (Botfield and Wilson, 1988) were used for protein expression and functional studies. The expression plasmids pK95/ΔAH/WT MelB_St/CHis₁₀ (Guan et al., 2011) and pK95/ΔAH/D59CMelB_St/CHis₁₀ (Ethayathulla et al., 2014) were used for constitutive expression.

Cell growth for the large-scale production of WT MelB_St or D59C MelB_St was carried out in E. coli DW2 cells (Ethayathulla et al., 2014; Pourcher et al., 1995). Briefly, MelB_St purification by cobalt-affinity chromatography (Talon Superflow Metal Affinity Resin, Takara) after extraction by 1.5% UDM. MelB_St protein was eluted with 250 mM imidazole in a buffer containing 50 mM NaPi, pH 7.5, 200 mM NaCl, 0.035% UDM, and 10% glycerol, and further dialyzed to change the buffer conditions accordingly.

The Micro BCA Protein Assay (Pierce Biotechnology, Inc) was used to determine the protein concentration.

MelB_St-mediated α-NPG downhill transport was detected by the release of the intracellular α-NPG hydrolytic product, p-nitrophenol, from E. coli DW2 cells expressing high-turnover number α-galactosidase and recombinantly expressed MelB_St . E. coli DW2 cells carrying the constitutive expression plasmid for the WT MelB_St or D59C MelB_St uniporter mutant in LB media containing 100 mg/L ampicillin were grown in a 37 °C shaker as described (Guan et al., 2011). The overnight cultures were diluted fivefold into fresh LB broth and 100 mg/L ampicillin and then shaken at 30 °C for 4 hr. The expression of α-galactosidase was induced by adding 5 mM melibiose for 1 hr before harvesting the cells for the transport assay. The melibiose-induced cells were washed with 100 mM KP_i, pH 7.5, three times to remove the remaining melibiose and Na⁺, and adjusted to A₄₂₀ of 10 (∼0.7  mg proteins/ml) in the assay solution of 100  mM KP_i, pH 7.5, 10 mM MgSO₄, and 1 mM DTT in the absence or presence of 20 mM NaCl or LiCl. The cells under each condition were equilibrated at a 30 °C incubator for 10 min before the α-NPG transport assay, which was initiated by mixing 0.5 mM label-free α-NPG and incubating for 60 min. A 100 µL cell aliquot at the given time point of 0, 1, 5, 10, 20, 30, 60 min was quenched with 900 µL 0.33 M Na₂CO₃, followed by a centrifugation at 10,000 × g for 5 min to collect the clarified supernatant for absorbance measurements. The extracellular p-nitrophenol released from the intracellular hydrolytic product of the translocated α-NPG, which was supplied in the extracellular environment, was measured at 405 nm by a UV spectrometer. The concentration of p-nitrophenol is estimated based on its molar extinction coefficient of 18,000 M^–1cm^–1.

The raffinose active transport was carried out by [³H]raffinose uptake with DW2 cells expressing MelB_St without the induction of α-galactosidase as described for the melibiose transport assay (Guan et al., 2011). Briefly, the cells expressing the WT MelB_St or D59C MelB_St uniporter mutant, or without a plasmid, were mixed with 2 µL of 25 mM [³H]raffinose (specific activity 10 mCi/mmol) to 50 µL of cells final raffinose concentration at 1 mM in the absence or presence of 50 mM NaCl or LiCl, and the transport reaction was quenched at the given time points and followed by a fast filtration.

All ITC ligand-binding assays were performed with the TA Instruments (Nano-ITC device) as described (Hariharan and Guan, 2017), which yields the exothermic binding as a positive peak. The MelB_St was dialyzed overnight with assay buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol, and 0.035% UDM. The ligands are prepared by dissolving in the same batch of dialysis buffer for buffer matching. In a typical experiment, the titrand (MelB_St) placed in the ITC Sample Cell was titrated with the specified titrant raffinose or α-methyl galactoside (placed in the Syringe) in the assay buffer by an incremental injection of 2 or 2.5 μL aliquots at an interval of 250 or 300 s at a constant stirring rate of 250 rpm (nano-ITC). MelB_St protein samples were buffer-matched to the assay buffer by dialysis. The normalized heat changes were subtracted from the heat of dilution elicited by the last few injections, where no further binding occurred, and the corrected heat changes were plotted against the mole ratio of the titrant to the titrand. The values for the binding association constant (K_a) were obtained by fitting the data using the one-site independent-binding model included in the NanoAnalyze software (version 3.7.5). The dissociation constant (K_d)=1/K_a.

The D59C MelB_St was dialyzed overnight against the sugar-free dialysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.035% UDM, and 10% glycerol), concentrated with Vivaspin column at 50 kDa cutoff, and stored at –80 °C. A phospholipid stock solution of 20 mM was prepared by dissolving the E. coli Extract Polar (Avanti, 100600) with a dialysis buffer containing 0.01% DDM. The protein sample was diluted to a final concentration of 10 mg/ml with the same sugar-free dialysis buffer, supplemented with phospholipids at 3.6  mM and 30 mM of melibiose or α-MG, 40 mM raffinose, or 6 mM of α-NPG in DMSO solution. Crystallization trials were conducted using the hanging-drop vapor-diffusion method at 23 °C by mixing 2 μL of protein with 2 μL of reservoir solution. Crystals from D59C MelBSt protein with the melibiose, α-MG, or α-NPG appeared against a reservoir consisting of 100 mM Tris-HCl, pH 8.5, 100 mM NaCl₂, 50 mM CaCl₂, and 32–35% PEG 400. For the raffinose-containing sample, the crystals were collected from 100 mM Tris-HCl, pH 8.5, 50 mM CaCl₂, 50 mM BaCl₂, and 32.5% PEG 400. All crystals were frozen in liquid nitrogen within 2 weeks and tested for X-ray diffraction at the Lawrence Berkeley National Laboratory ALS beamlines 5.0.1 (for the melibiose, α-MG, and α-NPG-containing complex datasets) or 5.0.2 (for the raffinose-containing datasets) using the remote data collection method.

ALS auto-processing XDS or DIALS programs output files were further reduced by AIMLESS in the ccp4i2 program for the structure solution (Potterton et al., 2018). The statistics in data collection are described in Supplementary file 1.

The structure determination was performed by the Molecular Replacement method using the α-NPG-bound D59C MelB_St mutant structure [PDB ID 7L17] as the search template, followed by rounds of manual building and refinement to resolutions of 2.60 Å, 3.05, 3.45, or 3.68 Å for structure with α-NPG, melibiose, raffinose, or α-MG, respectively, in Phenix (Liebschner et al., 2019). The model building and refinement were performed in Phenix and Coot (Casañal et al., 2020), respectively. The structures were modeled from positions 2–453 or 455, respectively, without gaps, and the missing side chains due to density disorder, as well as the Ramachandran assessment, are listed in Supplementary file 5.

One strong positive density, with varying sizes and shapes, was observed in the difference maps of each of the four structures. The size and shape matched the sugars that co-crystallized, and the docked sugar molecules fitted well with the densities. The sugar refinement restraints were generated from SMILES using the ELBOW program in Phenix (Liebschner et al., 2019). To the 2.60 Å α-NPG-bound map, five water molecules were modeled. In addition, a PEG molecule (ligand ID 1PE) was also modeled to a strong positive density with a sausage shape aligning with helix IX. To the melibiose-bound structure, two water molecules were added.

An in-solution HDX-MS experiment was performed to study the substrate-induced structural dynamics of MelB_St. As described (Hariharan et al., 2024b), the labeling, quenching, lipid removal, and online digestion were achieved using a fully automated manner using an HDx3 extended parallel system (LEAP Technologies, Morrisville, NC; Hamuro et al., 2003; Hamuro and Coales, 2018). MelB_St was prepared at 50.0 μM in a Na⁺-free buffer (25 mM Tris-HCl, pH 7.5, 150 mM choline chloride, 10% glycerol, 0.035% UDM in H₂O), either in the absence of a substrate (apo) or in the presence of 100 mM melibiose, 100 mM NaCl, or both. The hydrogen/deuterium exchange reaction, as described previously (Hariharan et al., 2024b). Briefly, aliquots of 4 µl of each sample were diluted 10-fold into the labeling buffer (25 mM Tris-HCl, pD 7.5, 50 mM choline chloride, 10% glycerol, 0.035% UDM in D₂O), without or with 100 mM of melibiose, Na⁺, or both. Labeled samples were incubated in D₂O buffer at 20 °C for multiple time points (30 s, 300 s, and 3000 s) in triplicate, and non-deuterated controls were prepared similarly, except that H₂O buffer was used in the labeling step.

At each designated time point, the reaction was quenched by adding an equal volume of ice-cold quench buffer (6 M urea, 100 mM citric acid, pH 2.3 in H₂O) for 180 s at 0 °C and immediately subjected to a lipid filtration module integrated on the LEAP PAL system. After incubation of a 60 s with ZrO2 particles, the LEAP X-Press then compressed the filter assembly to separate proteins from the ZrO2 particles-bound phospholipids and detergents. The filtered protein sample was injected into a cool box for online digestion and separation.

LC/MS bottom-up HDX was performed using a Thermo Scientific Ultimate 3000 UHPLC system and Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer. Samples were digested with a Nepenthesin-2 (Affipro, Czech Republic) column at 8 °C and then trapped in a 1.0 mm x 5.0 mm, 5.0 µm trap cartridge for desalting over 180 sec. The resulting peptides were then separated on a Thermo Scientific Hypersil Gold, 50x1 mm, 1.9 µm, C18 column with a gradient of 10 % to 40 % gradient (A: water, 0.1% formic acid; B: acetonitrile, 0.1% formic acid) for 15 minutes at a flow rate of 40 µL/min. A pepsin wash was added in between runs to minimize the carryover.

A nonspecific digested peptide database has been created for MelB_St with a separate MS/MS measurement of non-deuterated samples as described (Hariharan et al., 2024b). Digested peptides from undeuterated MelB_St protein were identified on the orbitrap mass spectrometer using the same LC gradient as the HDX-MS experiment. Using the Thermo BioPharma Finder software (v 5.1), MS2 spectra were matched to the MelB_St sequence with fixed modifications.

A total of 146 or 150 peptide assignments (with confident HDX data across all labeling times) were confirmed for MelB_St samples, resulting in 86–87% sequence coverage. The MS data were processed using the Sierra Analytics HDExaminer software with the MelB_St peptide database. Following the automated HDX-MS analysis, manual curation was performed. Upon the completion of the data review, a single charge state with high-quality spectra for all replicates across all HDX labeling times was chosen to represent HDX for each peptide. Differential HDX data were tested for statistical significance using the hybrid significance testing criteria method with an in-house MATLAB script, where the HDX differences at different protein states were calculated (ΔD=D_Holo DAp_o). Mean HDX differences from the three replicates were assigned as significant according to the hybrid criteria based on the pooled standard deviation and Welch’s t-test with p<0.05. The statistically significant differences observed at each residue (ΔD_{Mel - Apo} < |0.186|, ΔD_(Na+) - Apo < |0.224|, or ΔD_Na(+)Mel - Apo < |0.175|) were used to map HDX consensus effects based on overlapping peptides onto the structure models.

All experiments were performed two to four times. The average values were presented with standard errors. An unpaired t-test was used for statistical analysis. For the relative D%, the data were transferred to log values prior to the unpaired t-test.

Pymol (3.1.5.1) (Schrodinger, 2013) and UCSF ChimeraX (Pettersen et al., 2021) were used to generate all graphs. The program Origin 2024 was used to plot the ITC curves and transport data.

The crystal structure of the α-NPG-bound D59C MelB_St at a resolution of 2.6 Å was replaced with a melibiose molecule according to the melibiose-bound structure, and the D59C was mutated back to Asp. Both Asp59 and Asp55 residues were set in the deprotonated state. Three systems, including the apo, melibiose-bound, and melibiose- and Na⁺-bound states, were generated by embedding them in a POPE:POPG (7:2) lipid bilayer created using the CHARMM-GUI (Wu et al., 2014) web server. The lipid bilayer was capped with a 25 Å water box on each side, and ~0.15 M NaCl was added to neutralize the system charge and mimic the ionic strength of physiological conditions. The resulting system has ~120,000 atoms, with a periodic boundary condition of ~110 × 110×125 (Å). The ff14SB (Maier et al., 2015), lipid17 (Skjevik et al., 2016), and GLYCAM (Group, 2005) force fields were employed to treat the protein, lipid, and melibiose, respectively. The TIP3P (Jorgensen et al., 1983) water model was used for all water molecules.

After system assembly, energy minimization was carried out with harmonic restraints (1000 kJ/mol/Ų) on protein and lipid heavy atoms for 40,000 steps. This was followed by 200 ps of equilibration in the constant NVT ensemble at 300 K and 1 ns of NPT equilibration with gradually decreased force constants in the harmonic restraints. Five independent replicas of production simulations in the constant NPT ensemble were initiated from different snapshots in the NVT trajectory. For each replica, a trajectory was propagated for ~400 ns at 300 K and 1 atm. The total sampling time for each system was ~2 μs. The temperature was controlled with a Langevin thermostat (Sindhikara et al., 2009) with a friction coefficient of 1 ps^–1 and pressure with a Berendsen barostat (Uberuaga et al., 2004) using anisotropic scaling with a relaxation time of 1 ps. Long-range electrostatics were treated with the Particle Mesh Ewald (PME) method (Darden et al., 1993) (tolerance 5×10^–4), and van der Waals interactions were truncated using a 12 Å cutoff distance. A 2 fs timestep was used throughout all simulations. The SHAKE algorithm (Ryckaert et al., 1977) was employed to constrain the lengths of all hydrogen-containing covalent bonds. All MD simulations were performed with the AMBER24 software package (Case et al., 2023).

Water-1 occupancy identified in the α-NPG-bound crystal structure was evaluated by monitoring interactions of water molecules with four coordinating residues. For each frame, a water molecule was defined as located in the sugar-binding pocket if the distances from its oxygen (O) atom to a few neighboring residues and the melibiose molecule satisfied the following rules. First, four pairs of heavy atoms on the melibiose molecule and neighboring residues were defined: O5 on the galactosyl ring and NE2 on Gln372, O5 and OG1 on Thr373, O6 and NE2 on Gln372, as well as O6 and OG1 on Thr373. Second, for each pair of the above-defined heavy atoms, the distance between the water oxygen atom (O) and each of the two heavy atoms in the pair was calculated. Third, out of all four pairs, if at least in one of them both distances were within 4 Å and at least one distance was below 3.5 Å, the water molecule was defined as occupying the sugar-binding site. The water-1 occupancy in the sugar-binding site was then computed as the fraction of frames containing at least one occupying water molecule over the total number of frames in all replicas of the trajectories.

The side-chain heavy-atom root-mean-square fluctuation (RMSF) of all residues in the sugar- and cation-binding pockets was determined using CPPTRAJ.