Figures and data
Local resolution maps calculated in cryoSPARC.
The maps are colored from blue to red, with the higher resolution in darker blue. (A) The local resolution map of the unliganded form of the FnSiaQM-nanobody complex. The interior of the FnSiaQM is well-ordered. The maps are displayed at an r.m.s of 1.1. (B) The local resolution map of the Neu5Ac bound form. The insert depicts the location of Neu5Ac (center). The density for Neu5Ac is very well defined and the local resolution is about 2.6 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 ligand bound 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) Ribbon diagram showing the superposition of the HiSiaQM, PpSiaQM and FnSiaQM (liganded and unliganded) structures. The positions of the known Na+ ions and metal ion (M) are inspheres, and the position of Neu5Ac is shown in ball and stick. (C) 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 connected 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.
Binding Site of 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 circles, black squares, white triangles, black triangles, asterisks, and valinomycin, it 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. (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 Oο. (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. 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.
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).
Superposition of SiaQM structure from F. nucleatum, H. influenzae and P. profundum with respective bound nanobody or megabody. The structures were in inward-open conformation.
Cryo-EM workflow and analysis of the FnSiaQM protein. A) and B) a detailed workflow outlining the steps involved in cryo-EM image acquisition and processing, leading to the generation of both the bound and Apo structures of the FnSiaQM protein, respectively. The selected 2D class average utilized for ab initio reconstructions is depicted, and the optimal 3D reconstructions serve as reference models for subsequent non-uniform refinement. Masks generated using RELION 3.0 were applied, and the resulting maps underwent iterative rounds of local refinement. Fourier shell correlation (FSC) curves for the final 3D reconstructions of the bound and Apo structures of the FnSiaQM protein are presented.
SiaQM protein sequence alignment. SiaQM protein sequences from Photobacterium profundum, Haemophilus influenzae, and Fusobacterium nucleatum. Protein sequences were aligned using ESPript 3.
A) Na1 site coordination and its interactions with neighboring amino acids; B) Na2 site coordination and its interactions with neighboring amino acids; C) Metal binding site coordination and its interactions with neighboring amino acids.
A and B. Density of two lipids (Lipid1 and Lipid2) was observed in the scaffold side of FnSiaQM (contour of 1.0 r.m.s. in PyMol). C. Electrostatic potential visualization of FnSiaQM. Neu5Ac density in the binding pocket of FnSiaQM. ABPS colored in the range from −5 to +5.
