Crystal structure of D59C MelBSt in complex with α-sugars or sugar analogs.

The identities of the bound sugars are indicated at the top. Upper row: Cartoon representation of the structures of D59C MelBSt bound with α-nitrophenyl galactoside (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: the sugar-binding pockets. 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. (a) Isomesh map of the α-NPG-binding site, contoured at a level of σ =1.5. Dashed lines indicate distances within hydrogen-bonding or salt-bridge interactions (Å). W1, water molecule 1 in red. The helices are labeled with Roman numerals.

Binding of melibiose, α-MG, and raffinose.

All residues within a 5-Å distance to the bound substrates were shown in sticks. 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. 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′) are labeled on the melibiose molecule. Trp342 was removed for clarity in panels a, b, and d.

Binding affinity determination via ITC.

The binding measurement of α-MG or raffinose to the WT MelBSt was performed with a NanoITC calorimeters (TA Instruments) at 25 °C as described in Methods. 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). (a) α-MG. (b) Raffinose.

HDX-MS.

HDX experiments on WT MelBSt in 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 ΔDs, calculated from paired conditions for position 2-470, were plotted against overlapping peptides for each time point and the total uptake. Black, cyan, and purple bars, the deuterium uptake at 30, 300, 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 triplicates. Regions not covered by labeled peptides were excluded. Dashed lines, the global threshold values calculated from each dataset. (b) Mapping onto the 3D structure of the melibiose-bound state. 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 MelBSt is indicated by a gray bar. Each peptide or overlapping peptide was indicated by arrows pointing to the start of the peptide. The helices I, V, VIII, and IX, as well as the C-terminal helix (lid), are labeled individually. Mel, melibiose.

Allosteric effects on deuterium uptake by remote binding of Na+.

The time course of percentage deuterium uptake was plotted against the labeling time at 0, 30, 300, and 3000 sec for the representative peptides that cover the residues responsible for the specificity of sugar or cation binding in MelBSt. The peptide sequences are shown for each panel. *, statistically significant; **, statistically highly significant. Helices I and V (sugar-binding only), II (cation binding only), and IV (both), were shown in tube representation. Residues involved in sugar binding are represented by sticks and highlighted in magenta (helix I), blue (helix IV), and red (helix V). Residues involved in cation binding are highlighted in black. Labels in the time course and on the model are color-coded, except for the stick representing Thr121, which is black. Helices are labeled with Roman numerals and positioned at the beginning of each helix.