Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na+-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. Collectively with the available outward-facing sugar-bound structures, both the outer and inner barriers were localized. The N- and C-terminal residues of the inner barrier contribute to the sugar selectivity pocket. When the inner barrier is broken as shown in the inward-open conformation, the sugar selectivity pocket is also broken. The binding assays by isothermal titration calorimetry revealed that this inward-facing conformation trapped by the conformation-selective Nb exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for the substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is also supported by molecular dynamics simulations. Furthermore, the use of this Nb in combination with the hydron/deuterium exchange mass spectrometry allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.


Contents:
-Supplemental Table sTable 1 Cryo-EM data collection and structure determination statistics.sTable 2 HDX reaction and labeling details. sTable 3. E. coli strains and plasmids used in this study.

No addition In the presence of Nb725_4
In the presence of Nb725 sFig.3. Nb effects on substrate/ligand binding.All ITC binding measurements at 25 °C and curve fitting were described in the Methods.The binding measurements of melibiose, α-NPG and EIIA Glc were conducted in buffer, 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.01% DDM, and 10% glycerol.The Na + binding measurements were conducted in 20 mM Tris-HCl, pH 7.5, 100 mM choline-Cl, 0.01% DDM, and 10% glycerol as described in (Hariharan, 2017).All proteins and ligand solutions were buffer-matched.MelBSt complexed with Nb725_4 or Nb725 were prepared by mixing them at a 1:2 molar ratio.The thermograms of each titration (black) were plotted by bottom/left (x/y) axes and the binding isotherm and fitting using one-site independent-binding model presented by top/right (x/y) axes.The corresponding controls by titrating each ligand into the protein-free buffer (cyan) were presented below at an identical scale.

Fwd
Nb725_4 generated by CDR grafting.(a) All CDR regions are indicated by boxes.The sequences of TC-Nb4 and MelBSt Nb725 were colored in black and red, respectively.The sequence of hybrid Nb725_4 was colored in black and red from the two parents Nbs.The Nb725_4 binding residues were highlighted in underlined bold.(b-d) ITC measurements of Nbs binding to the NabFab.The Kd values for Nb725_4 and the Anti-Fab Nb binding to NabFab were presented as the mean ± sem and the number of tests = 2. N, the binding stoichiometry number.The interaction of the parent Nb725 to the NabFab was too weak to be fit accurately.(e) Isolation of the MelBSt/Nb725_4/NabFab/anti-Fab Nb complex.MelBSt proteins were reconstituted into the *** *****.*:*******:*:*********.***:*******

sFig. 2 .
Effects of substrate/ligand binding on the Nbs binding.All ITC binding measurements at 25 °C and curve fitting were described in the Methods.The purified Nb725_4, Nb725, MelBSt, and EIIA Glc protein samples were dialyzed against an identical buffer containing 100 mM NaCl.MelBSt at a concentration of 35 µM was placed in the sample cells and titrated with Nb725_4 or Nb725, respectively.The thermograms of each titration (black) and the corresponding control by titrating Nbs into the protein-free buffer (light gray) were plotted by the bottom/left (x/y) axes.The binding isotherm and fitting using a one-site independent-binding model presented by top/right (x/y) axes.(a) Binding of Nb725_4 to the Na + -bound MelBSt in the absence or presence of melibiose (80 mM), α-NPG (5 mM), or EIIA Glc (2:1 ratio to MelBSt).(b) Binding of Nb725 to the Na + -bound MelBSt in the absence or presence of melibiose (80 mM), α-NPG (5 mM), or EIIA Glc (2:1 molar ratio to MelBSt).
(a) Titration of melibiose to MelBSt or a given MelBSt/Nb complex at 80 µM for MelBSt.(b) Titration of α-NPG binding to MelBSt a given MelBSt/Nb complex at 50 µM for MelBSt.(c) Titration of Na + to MelBSt or a given MelBSt/Nb complex at 80 µM for MelBSt.(d) Titration of EIIA Glc to MelBSt or a given MelBSt/Nb complex at 50 µM for MelBSt.For the titration into the MelBSt/Nb complexes, melibiose and α-NPG in the assay solutions were increased from 10 mM to 80 mM or 1 mM to 10 mM.The Na + at 3 mM and EIIA Glc at 0.5 mM were applied for titrations.sFig.4. CryoEM data process.The strategy for the reconstruction and refinement was outlined and the details were presented in panels b-c.sFig. 5. GSFSC resolution and 3dFSC.The resolution of map and particle orientation distribution was assessed by cryoSPARC program using default setting parameters.sFig.6. Evaluation of map and models.(a) The local resolution.The map half_A and half_B files produced by the cryoSPARC Local Refinement program were used to calculate the Local Resolution Map by Phenix and displayed by UCSF ChimeraX using the defined color key.(b) Qscores.The auto-sharpened map generated by Phenix under the default setting was used as the main map for the model building and also for Q-score calculations by MapQ program in UCSF Chimera against the final structure at a sigma level of 0.4.The results of the Q scores for each residue were color-painted on the structure using the given color key.The bound Na + was shown as a sphere.The expected score at a resolution of 3.29 Å is 0.44.(c) Density mapped on the MelBSt transmembrane helices.The auto-sharped map by Phenix was used to calculate the isomesh maps for each transmembrane helix at a sigma level of 10 and carving of 1.8.sFig.7. NabFab comparison.(a & b) NorM complexed with anti-NorM Nb17_4/NabFab/anti-NabFab Nb [PDB ID 7PHP] was superimposed with MelBSt complexed with Nb725-4 and NabFab based on the H chains of NabFab in the two structures.Nb725-4 and NorM-Nb17_4 were colored in the rainbow.(c) The NabFab in the NorM complex [PDB ID 7PHP] was superimposed with that in MelBSt complex.(d) The NabFab/Nb in the NorM complex was superimposed with that in MelBSt complex based on their H chains. Green, MelBSt complex; yellow, NorM complex. of Nb725m_4 and MelBSt.(a) Nb725_4 bound to the N-terminal domain of MelBSt.The contact between Nb CDR-1 and CDR-2 and MelBSt loop4-5, and loop6-7 contributed to the major interactions.(b) The surface electro potential map was generated in the UCSF Chimera X program.The inward-open cavity is indicated.(c) The binding interface.All residues with a buried solvent-accessible surface area >15 Å 2 or polar interaction at a distance <3.5 Å were selected by the UCSF Chimera X program were highlighted in sticks.The salt-bridging and hydrogen-bonding interactions were highlighted by the dashed line.The Nb CDRs and epitope are indicated.sFig.9. Complex of MelBSt with Nb725 and EIIA Glc .Samples containing MelBSt (black curve), MelBSt with Nb725 (red curve), or with Nb725 and EIIA Glc (blue curve), were prepared and analyzed by gel filtration chromatography in a combination of the SDS-15%PAGE stained by silver nitrate.Mixture, the solution was prepared prior to the gel filtration chromatography containing all three proteins.1 µg of each protein was loaded as the control.sFig.10.MD simulations of the Na + binding at both inward-and outward-facing states.The equilibrated structures of Na + -binding site for the inward-and outward-facing MelBSt in the absence of melibiose.Distances between Na + and nearby residues' coordinating atoms are labeled in text and dashed lines.(a) Na + binding in the inward-facing conformation.(b) Na + binding in the outward-facing conformation.(c) Probability distribution of the distance between the bound Na + to all ligands.Blue, inward-facing conformation; green, outward-facing conformation. .Galactose-binding pocket in the outward-facing crystal structure [PDB ID 7L17].All polar interactions between the bound a-NPG and D59C MelBSt were indicated by dash lines and the pocket was 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 indicated.Eight N-terminal residues (Lys18, Asp19, Ile22, Tyr26, Tyr120, Asp124, Trp128, and Arg149), especially the four charged residues, form the multiple polar interactions with one surface of the galactopyranosyl moiety.Five C-terminal residues (Trp342, Gln372, Thr373, Val376, and Lys377) form a non-specific barrier without a strong polar interaction The alignment of the sugar-bound outward-facing structure (7L17, green) with the Na + -bound inward-facing structure (blue) were carried out in Pymol or Coot programs.The rmsd values reported from Pymol program were either based on default settings with outlier rejection or using all atoms after removing unmatched residues.(a) Superposition of both structures.(b) Full-length alignment based on positions 2-200.(c) Full-length alignment based on positions 231-332.(d) Focused alignment of positions 2-200.(e) Focused alignment of positions 231-432.(f) Helices I, II, and IV isolated from the focused alignment of positions 2-300 in d.The major binding residues for Na + and α-NPG were highlighted.(g) Helices VII-VIII and the extended loops isolated from the focused alignment of positions 219-432.(h) Helices X-XI and the extended loops isolated from the focused alignment of positions 219-432.sFig.13.Membrane topology.(a) The sugar-bound outward-facing structure (PDB ID 7L17).(b) The Na + -bound inward-facing structure.The full-length MelBSt is illustrated by transmembrane topology based on both structures.The residue positions for each helix are indicated.The N-and C-terminal transmembrane helices are colored green and orange for the outward-facing conformation, respectively, as well as blue and light blue for the inward-facing conformation, respectively.The peripheral helices are colored in light yellow.Blue ball, Na + ; yellow hexagons, galactoside.The red triangle indicates the solvent-access paths.Grey lines, loops; dashed lines, un-resoled loops.Broken circles, either cation-specificity pocket or sugarspecificity pocket.sFig.14.Histograms of deuterium uptake time courses.The deuterium uptake time courses of the groups I&II presented in the Fig. 7 were replotted in the histogram.Error bar, sem; test number, 3.