Figures and data
Architecture of FnSiaQM with nanobody. (A and B) Cryo-EM maps of FnSiaQM unliganded and Neu5Ac bound at 3.2 and 3.17 Å, respectively. The TM domain of FnSiaQM is colored using the rainbow model (N-terminus in blue and C-terminus in red). The nanobody density is colored in red. The density for modeled lipids is colored in tan and the unmodelled density in gray. The figures were made with Chimera at thresholds of 1.2 and 1.3 for the unliganded and Neu5Ac-bound maps. (C and D) The cytoplasmic view of apo and Neu5Ac bound FnSiaQM, respectively. Color coding is the same as in panels A and B. The density corresponding to Neu5Ac and sodium ions are in purple. The substrate binding sites of apo and Neu5Ac bound FnSiaQM are shown with key residues labeled. The density (blue mesh) around these atoms was made in Pymol with 2 and 1.5 σ for the apo and the Neu5Ac structures, respectively, with a carve radius of 2 Å.
Details of cryo-EM data collection, processing, refinement and built model.
FnSiaQM-Nanobody complex with Neu5Ac bound form.
(A) A cartoon representation of the FnSiaQM-Nanobody complex structure. The SiaQM polypeptide has been colored in rainbow with the N-terminus starting in blue and the C-terminus ends in orange. The nanobody is shown in cartoon representation and in red color. Two of the modeled sodium sites are shown as purple spheres. A ribbon representation of the unliganded structure of FnSiaQM is superposed in grey. The superposition reveals that the overall structures are similar. (B) Cartoon representation of the FnSiaQM structure bound to Neu5Ac. In shades of blue are the helices that form the scaffold domain. In gray is the connecting domain. In olive, orange, and red is the elevator domain. HP1 and HP2, the two helix-loop-helix motifs, are in red and marked. The positions of the known Na+ ions and metal ion (M) are in spheres, and the position of Neu5Ac is shown in ball and stick.
Ion binding sites in FnSiaQM
(A) Close-up view of the Neu5Ac and the two Na+ and metal binding sites. In red is the HP1 helix-loop-helix. In green is the HP2 helix-loop-helix. In blue is helix 5 A, the purple spheres are the two Na+ ion binding sites and metal binding site (M), and the bound Neu5Ac is shown in ball and stick. (B-D) Density and interaction details of the Na1, Na2, and M sites, respectively. The figures are made with a contour of 1.0 r.m.s. in PyMol.
Proteoliposome transporter assays for FnSiaQM.
(A) Schematic diagram showing the experimental setup. First, FnSiaQM is incorporated into proteoliposome in the presence of internal K+. Then, valinomycin (val) is added to induce the efflux of K+ down its concentration gradient, imposing an artificial membrane potential. To start transport measurement, FnSiaP, [3H]-Neu5Ac and Na+-gluconate are added in the extraliposomal environment. (B) Time course of Neu5Ac uptake into proteoliposomes reconstituted with FnSiaQM. In black circle, black square, white triangles, black triangle, asterisk, are with conditions, where valinomycin was added to facilitate K+ movement before transport. Ethanol was added instead of valinomycin as a control in the white circle, white square, and white triangle. In white square, black square, white triangle, black triangle, 10 mM Na+-gluconate was added together with 5 µM [3H]-Neu5Ac and 0.5 µM FnSiaP; in white triangle and black triangle proteoliposomes are prepared without internal K+; in black asterisk transport is measured in the presence of 10 mM Na+-gluconate, 5 µM [3H]-Neu5Ac and in the absence of FnSiaP. Uptake data were fitted in a first-order rate equation for time course plots. Data are means ± s.d. of three independent experiments.
Validation of Neu5Ac binding pocket in FnSiaQM transporter.
(A) The fit of the modeled Neu5Ac into the density, contoured to 0.9 * r.m.s. The figure also shows the fit to the density of the residues that interact with Neu5Ac and the distances to key residues discussed in the manuscript. (B) The interactions of Neu5Ac with side chains of interacting residues. Ser300 Oγ is 2.8 Å from the C1-carboxylate oxygen of Neu5Ac, while the main chain NH of residue 301 is 2.6 Å away. The other close polar side chain is that of Ser345γ, which is 3.3 Å away. The Neu5Ac O10 is 2.9 Å from Asp521 Oδ. (C) Transport of Neu5Ac in 15 min. Transport was started by adding 5 µM [3H]-Neu5Ac, 10 mM Na+-gluconate, and 0.5 µM FnSiaP to proteoliposomes in the presence of valinomycin (expected membrane potential, ΔΨ, −117.1 mV). Data are expressed as nmol/mg prot/15 min ± s.d. of three independent experiments. (D) The cavity of Neu5Ac is exposed from the cytoplasmic side. The cavity is large and extends into the cytoplasmic side. The black arrow shows the methyl group with an extra hydroxyl in Neu5Gc.
Comparison of Neu5Ac binding pocket
(A) The interactions of Neu5Ac with the substrate binding protein SiaP from F. nucleatum (PDB-ID 4MNP). The C1 and the C8 carbon atoms are labeled to show the different ends of the nine-carbon sugar. (B) the interaction of Neu5Ac with the SSS-type Neu5Ac transporter (PDB-ID 5NV9). (C) The interaction of fumarate with the dicarboxylate transporter VcINDY (PDB-ID 6OKZ).
