Functional characterization of the hybrid Nb725_4.

(a) In vivo two-hybrid interaction assay. The test was performed in E. coli DH5α cyaA cells on the maltose-containing MacConkey agar plate as described in Methods. When the T18 and T25 fragments encoded by two compatible plasmids were expressed separately, or only one fusion of either T25:MelBSt or Nbs:T18, no cAMP was produced, so no maltose fermentation was observed as shown in yellow colonies. The interaction of two hybrid proteins of T25:MelBSt and Nbs:T18 restored adenylate cyclase activities and cAMP production, which resulted in sugar fermentation. The irregular red colonies are typical of a positive two-hybrid test. (b) Nb inhibition of melibiose fermentation. Two compatible plasmids encoding MelBSt and Nb725 or Nb725_4 were transformed into E. coli DW2 cells [ΔmelBΔlacYZ] and plated on the MacConkey agar plate containing maltose (as the positive control) and melibiose (indicating melibiose 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. Purified Nbs and MelBSt protein samples were dialyzed against 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.01% DDM, and 10% glycerol. Exothermic thermograms 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 (ΔQ) 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

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

CryoEM-SPA.

The sample containing the wild-type MelBSt in lipids nanodiscs, the MelB- 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. MelB 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.

Na+-binding and sugar-binding pockets.

(a) Na+-binding pocket in the inward-facing conformation (PDB ID 8T60). The isomesh map 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. (b) Galactoside-binding pocket in the outward-facing conformation [PDB ID 7L17]. All polar interactions between the bound α-NPG and D59C MelBSt were indicated by dash lines and the pocket was also shown in surface representation in blue. The Na+-binding residues were highlighted by sticks and surface representation in pink. The cytoplasmic and periplasmic sides were labeled. (c) Superimposed co-substrate-binding pockets. The a-NPG-bound outward-facing structure [7L17] in b was aligned on the inward-facing structure based on the 2-200 region. The residues in the sugar- and cation- binding pockets, as presented in the outward-facing structure in b, were colored in black and labeled in blue in the inward-facing structure. 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 [PDB ID 8T60; 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 (colored yellow) in N- and C-terminal bundles were colored dark gray and cyan, respectively. (b&e) Cytoplasmic view. The charged network between the N- and C-bundles were colored in green and bright orange in panel b, or blue and light blue in panel e, respectively. The middle loop and the C-terminal tail were colored in red and the C-terminal helix panel b was set in transparent and missed in the inward-facing conformation. The charged residues were highlighted in sticks. Arg363 missed the sidechain in the inward-facing structure. (c&f) Periplasmic view. Loop11-12 was colored in red. The labels of the paired helices involved in the formation of either barrier were cycles in the same colors. The a-NPG was colored in yellow (b&c) and Na+ was shown in blue sphere (e&f).

MelBSt dynamics probed by HDX-MS.

MelBSt alone or complexed with Nb725_4 at a 1:2 ratio was 10-fold diluted into the labeling buffer in (25 mM Tris-HCl, pD 7.5, 150 mM NaCl, 10% glycerol, 0.01% DDM in D2O) and incubated at 20 °C for 30, 300, and 3000 sec, quenched with urea and citric acid pH 2.3. The lipids and detergent were removed by a filter system containing ZrO2 before performing LC/MS bottom-up HDX as described in Methods. Each sample was analyzed in triplicates. (a) Residual plots (DNb725_4-bound - Nb-free) against peptides for each time point and the sum of uptake. Black, cyan, and blue bars, the deuterium uptake at 30, 300, 3000 sec, respectively; the gray line, the sum of uptake from all three time points. Deprotection, ΔD (DNb725_4-bound – Nb-free) > 0; deprotection, ΔD < 0. Cylinders, helix positions at the N- and C-terminal domains, as well as loops. Yellow lines, cytoplasmic middle, and C-terminal tail loops. ML, middle loop. (b) Mapping of the significant changes at 3000 sec time point on the outward-facing structure [7L17]. Any peptide of ΔD with P ≤ 0.05 and ΔD ≥ |0.3184| were treated as significant and positions were labeled in each time course plot. The representative peptides were also presented by uptake time courses using the percentage of deuterium uptake (D %) relative to the theoretic maximum number (the amino acid residue number minus two ending positions). The residue positions were labeled and the corresponding secondary structure was presented in the square bracket. The positions highlighted in the model were indicated in the uptake plots, either by a single peptide position or by a group of overlapping peptides as shown in the bracket. Error bar, sem; test number, 3. Other symbols were included in the figure.

Deuterium uptake 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 Nb725_4-bound structure [8T60] in the panel b, respectively. The peptide positions rwere labeled and the charged residues forming the inner barrier-specific salt-bridge network were indicated. In the Nb725_4-bound structure (b), the C-terminal tail was missing. The cartoon colored in dirtyviolet, the group I peptides with faster HDX rate; the cartoon colored in red, group II peptides.

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

Melibiose active transport begins at step 1 and proceeds via the red arrows around the circle, with one melibiose and one cation inwardly across the membrane per circle. Melibiose efflux transport begins at step 6 and proceeds via the black arrows around the circle, with one melibiose and one cation outwardly across the membrane per circle. Melibiose exchange begins at step 6, and also takes 4 steps involving steps 5 - 2 as highlighted in green color.