Fig 1.
Coupled D-gal:H+ cotransport by E. coli DgoT.
(A) Diagram illustrating transport by sialin (left) and a VGLUT (right). A vacuolar-type H+ pump provides the H+ electrochemical driving force for both activities, but sialin uses the pH gradient to drive electroneutral cotransport of sialic acid with H+ out of the lysosome whereas the VGLUTs rely on membrane potential to drive glutamate uptake by synaptic vesicles against a pH gradient. (B, C) Liposomes containing pyr at pH 7.5 were reconstituted with (+) or without (−) E. coli DgoT and added to reaction buffer at pH 5.5. gal, glu (both 10 mM), or buffer alone were added at t = 0. The fluorescence emission at 510 nm with excitation at 460 nm was normalized to excitation at 415 nm (the isosbestic point for pyr versus pH). (B) The representative change in fluorescence ratio shows that D-gal but not glu or buffer control reduce the lumenal pH of proteoliposomes reconstituted with DgoT but not empty liposomes. Bar graph (right) shows the average rate of fluorescence change for glu and buffer relative to gal (n = 6). ****p < 0.0001. (C) Traces (left) show representative fluorescence ratios of proteoliposomes with (above) and without DgoT (below), both formed at pH 7.2, before and after the addition of 10 mM D-gal (at arrow) in external solution at pH 7.5, with or without 0.2 μM val. Both internal and external solutions contained 5 mM K+. Scatterplot (right) shows the difference of fluorescence decay between the addition of gal (t = 0) and the end of recording (t ≈ 220). ***p < 0.001 by one-way ANOVA with Bonferroni correction. n = 9 for proteoliposomes with DgoT; n = 4 for liposomes (see S1 Fig). The numerical data underlying this figure are included in S1 Data. DgoT, D-galactonate transporter; gal, galactonate; glu, gluconate; pyr, pyranine; val, valinomycin; VGLUT, vesicular glutamate transporter; WT, wild type.
Fig 2.
The inward-open structure of DgoT shows a proton translocation pathway connected to the periplasm.
(A) Cylinder representation of inward-facing DgoT viewed parallel to the membrane plane and from the cytoplasm. Helices are numbered from the N to C terminus. Dotted line represents unstructured regions. (B) Overall view of inward-facing DgoT in cylinder representation shows the putative water tunnel (tan surface) with the surrounding residues shown as sticks. (C) A close-up view of the tunnel highlights the entrance and exit points. (D) Putative sites of protonation (Asp46 and Glu133) lie in a membrane-embedded pocket of polar residues buried within the NTD (crossed-eye stereo). The polar and charged residues composing the pocket are shown as sticks. Electron density is from a composite, simulated annealing “omit map” to eliminate model bias (1σ). Outer surface and cavities formed from interdomain contacts are shown in blue (NTD) and green (CTD). Asp46, aspartate-46; CTD, C-terminal domain; DgoT, D-galactonate transporter; Glu133, glutamate-133; ICH, intracellular helix; NTD, N-terminal domain.
Fig 3.
Functional residues coupled to proton or substrate transport.
(A) Representative fluorescence of proteoliposomes reconstituted with WT (blue) or mutant DgoT (D460N red, E133Q green) in response to addition of D-gal (darker color) or glu (lighter color) at t = 0 seconds. Bar graph (right) shows the average change in fluorescence at t = 90 seconds. Filled bars indicate galactonate, open bars gluconate (n = 11). ***p < 0.001; ****p < 0.0001. (B) Whole-cell uptake of radiolabeled 14C-galactonate by WT and mutant DgoT exogenously expressed in an E. coli DgoT knock-out strain. WT but not D46N or E133Q DgoT confer 14C galactonate uptake (n = 3). The numerical data underlying this figure are included in S1 Data. DgoT, D-galactonate transporter; gal, galactonate; glu, gluconate; ns, not significant; pyr, pyranine; WT, wild type.
Fig 4.
Properties of gal transport by DgoT.
(A) Uptake of 14C-gal by E. coli expressing DgoT shows inhibition by 1 mM gal but not by 10 mM glu or galac (n = 3). (B) Representative concentration dependence of 14C-gal uptake by cells expressing WT DgoT. The data from multiple experiments were each fit to the Michaelis-Menten equation and used to calculate the mean Km = 18 ± 4.1 μM and Vmax = 1.94 ± 0.42 nmol/(min × μg) (n = 3). (C) E. coli spheroplasts expressing WT DgoT show inhibition of gal uptake by the H+ ionophore nig (dissipating ΔpH) and val (dissipating Δψ), demonstrating electrogenic cotransport of H+ and gal. The numerical data underlying this figure are included in S1 Data. ****p < 0.0001; con, control; DgoT, D-galactonate transporter; gal, galactonate; galac, galactose; glu, gluconate; Km, Michaelis-Menten constant; nig, nigericin; val, valinomycin; Vmax, maximal rate; WT, wild type.
Fig 5.
The structures of DgoT include substrate-bound outward-facing occluded and apo-, inward-facing open conformations.
DgoT crystal structures shown in electrostatic surface potential representation. (A) A cross section through DgoT in the outward-facing occluded conformation shows galactonate (sticks) bound within the substrate recognition site (dashed white circle). The electrostatic potential of the periplasmic surface is shown below. (B) A cross section of DgoT in the inward-facing open conformation reveals an aqueous cavity with net positive surface charge. The electrostatic potential of the cytoplasmic surface is shown below. Both views highlight the positively charged binding site. DgoT, D-galactonate transporter.
Fig 6.
The outward-facing conformation contains D-galactonate occluded within the substrate recognition site.
(A) Close-up stereo view of the substrate recognition site. Residues Q164, Q264, S370, and N393 form hydrogen bonds (orange dashes) with the 5 hydroxyl groups in D-galactonate. Y44 and Y79 form hydrogen bonds, and Arg47 forms a salt bridge with the carboxyl group of D-galactonate. The Fo-Fc density map of D-galactonate (green mesh) was contoured at 3σ, and the 2Fo-Fc density map (gray mesh) of DgoT residues was contoured at 1σ. (B) The overall structure of DgoT in cylinder representation defines the views shown in panel A (black rectangle), panel C (purple), and panel D (orange). N- and C-terminal domains are shown in blue and green, respectively. ICH1 is shown in cyan and ICH2 is shown in bright green. D-galactonate (yellow) is shown in stick representation. (C) Hydrophobic gating residues F137 and W373 interact with the substrate while forming contacts between N- and C-terminal domains. N141 forms a cytoplasmic gate by hydrogen bonding with the backbone carbonyl of W373 and the hydroxyl of S377. (D) Tripartite interactions between TM1, TM5, and ICH1. This view is rotated 190° as indicated for clarity. (E) Cylinder representation of the N- and C-terminal domains separated and rotated 90° (left) and 90° (right) show the extent of structural change between inward-open (green) and outward-occluded (yellow) states. With substrate bound, TM7 bends approximately 20° to partially occlude the substrate from the periplasmic side. The helical discontinuity of TM4 in the outward-occluded state and of TM10 in the inward-open state are indicated by black arrows. Arg47, arginine-47; DgoT, D-galactonate transporter; Fo-Fc, difference map; ICH, intracellular helix; TM, transmembrane.
Fig 7.
Proposed mechanism for coupling H+ to substrate translocation.
Mechanism described counterclockwise from top left. In the outwardly oriented, occluded structure (top left), both Asp46 (D) and Glu133 (E) are protonated (H), liberating Arg47 (R) to interact with the anionic substrate (Dgal−) and transition from outward-facing to inward-facing conformation (bottom left). Release of substrate into the cytoplasm is captured in the inward-facing structure (bottom middle). Deprotonation of Glu133 could then enable the formation of a charge pair with Arg47 (bottom right) and so facilitate the transition of the empty transporter to the outward-facing state (top right). To account for electrogenic transport, we presume that Asp46 would also lose a H+ to the inside before this transition of the empty carrier. After reorientation to the outward-facing state, reprotonation of Asp46 and Glu133 (top middle) would allow Arg47 to interact with substrate (top left) and complete the cycle. We presume that the H+ tunnel to the periplasm occurs in the outward (as well as inward) orientation, before occlusion of the substrate, although we do not know whether proton(s) use this pathway or the main cavity to access the N-terminal polar pocket with Asp46 and Glu133. Arrows indicate the direction for inward uptake, although all the reactions are reversible. The VGLUTs lack an acidic residue in TM1 equivalent to Asp46 in DgoT and hence do not couple to H+. However, they contain a glu in TM4 equivalent to Glu133, and analogy with DgoT suggests that protonation of this site from the outside could allow the Arg in TM1 to bind neurotransmitter and facilitate transport. Protonation and deprotonation of a surrogate of Glu133 from the same side could account for allosteric activation of the VGLUTs by H+. This, in turn, could account for the efficient glu transport activity by synaptic vesicles, which is then inhibited by the higher pH in the synapse after vesicle fusion with the plasma membrane. Arg, arginine; Asp, aspartate; Dgal, D-galactonate; DgoT, D-galactonate transporter; Glu, glutamate; TM, transmembrane; VGLUT, vesicular glutamate transporter.