Functional characterizations of the hybrid Nb725_4.

(a) In vivo two-hybrid interaction assay. Two compatible plasmids encoding T25:MelBSt and Nb:T18 were transformed into E. coli DH5α cyaA cells and plated on the maltose-containing MacConkey agar plate as described in Methods. The irregular red colonies are typical of a positive two-hybrid test indicating protein-protein interactions. (b) Nb inhibition of melibiose fermentation. Two compatible plasmids encoding MelBSt and Nb725 or Nb725_4 were transformed into E. coli DW2 cells [∆melB∆lacYZ] and plated on the MacConkey agar plate containing maltose (as the positive control) and melibiose (for testing transport activity of MelBSt) as the sole carbon source. (c) [3H]Melibiose transport assay with E. coli DW2 cells. The cells transformed by two compatible plasmids encoding the MelBSt and Nb725 or Nb725_4 were prepared for [3H]melibiose transport assay at 0.4 mM (specific activity of 10 mCi/mmol) and 20 mM Na+ as described in Methods. The cells transformed with the two empty plasmids without MelB or Nb were the negative control. Inset, Western blot. MelBSt expression under the co-expression system was analyzed by isolating the membrane fractions. An Aliquot of 50 μg was loaded on each well and MelBSt protein was probed by anti-His antibody. (d) Nb binding to MelBSt by ITC measurements. As described in Methods, the thermograms were collected with the Nano-ITC device (TA Instrument) at 25 °C. Exothermic thermograms shown as positive peaks were obtained by titrating Nbs (0.3 mM) into the MelBSt-free buffer (gray) or MelBSt (35 mM)-containing buffer (black) in the Sample Cell and plotted using bottom/left (x/y) axes. The binding isotherm and fitting of the mole ratio (Nb/MelBSt) vs. the total heat change (f..Q) using one-site independent-binding model were presented by top/right (x/y) axes. The dissociation constant Kd was presented at mean ± SEM (number of tests = 6-7).

Nbs binding

CryoEM-SPA.

The sample containing the wild-type MelBSt in lipids nanodiscs, the MelBSt-specific Nb725_4, NabFab, and anti-Fab Nb at 1.5 mg/ml in 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl was prepared as described in the Methods. Images were collected using Titan Krios TEM with a K3 detector of S2C2, Stanford, CA. The particle reconstructions and modeling were performed as described in the methods. The final volume did not include the anti-Fab Nb during Local Refinement due to relatively poor densities. (a) The raw image after motion correction. (b) Representative 2D-Classes generated by CryoSPARC program. MelBSt in nanodiscs, Nb725_4, NabFab, and anti-Fab Nb can be easily recognized. (c) GSFSC resolution was calculated by cryoSPARC Validation (FSC) using two half maps generated by the CryoSPARC Local Refinement program. The number of particles used for the volume reconstruction was presented. (d) Particle distribution of orientations over azimuth and elevation angles generated by CryoSPARC Local Refinement program. (e&f) The structure of MelBSt/Nb725_4/NaFab complex. The volume (e) and cartoon representation (f) were colored by polypeptide chains as indicated. Nanodiscs were transparent and colored in light gray. Sphere and sticks in the panel f highlighted Na+ and its ligands.

Nb effects on MelBSt binding to sugar, Na+ and EIIAGlc

Na+-and sugar-binding pockets of MelBSt.

(a) Location of the Na+ binding site. The inward-facing cryoEM structure of the WT MelBSt was displayed in cylindrical helices with the cytoplasmic side on the top. One bound Na+ ion within the N-terminal helix bundle was shown in the blue sphere. (b) Na+-binding pocket. The isomesh map of the inward-facing conformation was created by the Pymol program using level 10 and carve of 1.8. The Na+ coordinates were shown in dash lines and interacting residues were shown in sticks. Qres, Q score for residue; Qsc, Q score for side chain. (c) Location of the galactoside-binding stie. The outward-facing x-ray crystal structure of D59C MelBSt mutant was displayed in cylindrical helices with the cytoplasmic side on the top [PDB ID 7L17]. One α-nitrophenyl galactoside (α-NPG) molecule was shown in the stick colored in yellow between the N-and C-terminal helix bundles. (d) Superimposed sugar-and cation-binding pockets. The a-NPG-bound outward-facing structure in c was aligned with the inward-facing cryoEM structure based on the 2-200 region. The residues in the sugar-and cation-binding pockets of the inward-facing cryoEM structure were colored in black and labeled in blue. D59C of the a-NPG-bound outward-facing structure is indicated in the parentheses. The a-NPG and Na+ were colored yellow and blue, respectively.

Barriers and sugar-binding pocket.

Outward-facing [PDB ID 7L17; left column] and inward-facing [right column] structures were used to prepare the figures. (a&d) Side view with cytoplasmic side on top. The inner and outer barriers were labeled. The sugar-specificity determinant pocket was highlighted in a dashed hexagon. The residues contributing to the bound a-NPG in N-and C-terminal bundles were colored in dark gray and cyan, respectively. (b&e) Cytoplasmic view. The charged network between the N-and C-terminal bundles were colored in green and bright orange in panel b, or blue and light blue in panel e, respectively. The C-terminal tail helix was set in transparent in panel b but disordered in the inward-facing conformation in panel e. The charged residues were highlighted in sticks. Arg363 side chain missed the sidechain in the inward-facing structure. (c&f) Periplasmic view. The paired helices involved in either barrier formation were highlighted in the same colored circles. The α-NPG was colored in yellow and Na+ was shown in blue sphere. The cytoplasmic middle loop, the C-terminal tail, and the periplasmic loop11-12 were highlighted in red.

MelBSt dynamics probed by HDX-MS.

(a) MelBSt peptide sequence coverage. The peptides of the deuterated MelBSt were determined based on the MelBSt peptide database that was generated by nonspecific digestions of non-deuterated MelBSt as described in the Methods. Peptides were confirmed in the HDX-MS experiment. Blue bar, the covering of each peptide. The amino-acid sequencing identification number should be -1 for each position due to the processed Met at position 1. The 10xHis Tag was included in the data analysis. (b) Residual plots (DNb725_4-bound - Nb-free) against the overlapping peptide numbers for each time point and the sum of uptake. MelBSt alone or bound with Nb725_4 in the presence of Na+ were used to carry out the HDX reactions as described in the Methods. Black, cyan, and blue bars, the deuterium uptake at 30, 300, 3000 sec, respectively; gray curve, the sum of uptake from all three time points. Deprotection, ΔD (DNb725_4-bound – Nb-free) > 0; protection, ΔD < 0. Each sample was analyzed in triplicates. Cylinders indicated the helices; the transmembrane helices were labeled in Rome numeral. The length of the cylinder does not reflect the length of corresponding helices but is estimated for mapping the deuterium-labeled overlapping peptides. The uncovered regions were not included. ML (cytoplasmic middle loop) and C-terminal tail including the His tag were colored in yellow. Dashed lines, the threshold.

Peptide mapping of HDX results.

HDX results were presented in Fig. 5. Any peptide of ΔD with P ≤ 0.05 and ∆D ≥ |0.3184| were treated as significant. The peptides with statistically significant differences at the 3000 sec time point were mapped on the outward-facing structure [PDB ID 7L17]. Inset, the deuterium uptake time course of representative peptides was plotted as a percentage of deuterium uptake relative to the theoretic maximum number (D%). The peptides, either as a single peptide or a group of overlapping peptides (in the parentheses), are labeled and indicated in the structure by the pink arrow in dashed lines. In the brackets, the corresponding secondary structure or loops. Error bar, sem; the number of tests, three. Other symbols were presented within the figure.

Dynamic regions of MelBSt.

The peptides that exhibited faster HDX rates (> 5% at 30 sec) were mapped on both the a-NPG-bound outward-facing structure [7L17] in panel a and the inward-facing cryoEM Nb725_4-bound structure in the panel b, respectively. All peptides were labeled and highlighted in a transparent gray box. The charged residues forming the inner barrier-specific salt-bridge network were highlighted in sticks and indicated by black arrows in dashed lines. In the Nb725_4-bound structure (b), the C-terminal tail was disordered. The cartoon colored in dirtyviolet, the group I peptides with faster HDX rate; the cartoon colored in red, group II peptides. The α-NPG was highlighted in yellow and Na+ was shown in blue sphere.

Stepped-binding model for the Na+/melibiose symport catalyzed by MelB.

Eight states are postulated including transient intermediates. In this reversal reaction, the cation binds prior to the sugar and releases after sugar release. Melibiose active transport or inflex begins at step [1] and proceeds via the red arrows clockwise around the circle, with one melibiose and one cation inwardly across the membrane per cycle. Melibiose efflux begins at step [6] and proceeds via the black arrows anti-clockwise around the circle, with one melibiose and one cation outwardly across the membrane per circle. Melibiose exchange begins at step 6, and only takes 4 steps involving steps [2 – 5] as highlighted in gray color. The low-sugar affinity inward-facing Na+-bound cryoEM structure represents the state after the release of sugar, as indicated by a dashed box.