Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site

Abstract

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na⁺ gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na⁺ ions. One Na⁺ binds to the conserved Na2 site, while the second Na⁺ binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na⁺ sites regulate N-acetylneuraminic acid transport.

Fig. 1

Overall architecture and the sialic acid binding site of SiaT. a Side-view of SiaT in the membrane plane. Transmembrane helices that coordinate with Neu5Ac and Na⁺ ions are depicted in colour, while the remaining helices are coloured in white. Neu5Ac is shown as grey spheres coloured by atom type and Na⁺ ions are shown as blue spheres. b Neu5Ac forms hydrogen bonds with Thr58 (Tm1), Thr63 (Tm1), Ser60 (Tm1) and Gln82 (Tm2) and a salt bridge with Arg135 (Tm3). Neu5Ac also forms water-mediated hydrogen bonds with Gln82 (Tm2), Asn247 (Tm6), Gln250 (Tm6) and Phe78 (Tm2). c Omit maps for Neu5Ac generated by removing respective ligands from the X-ray structure followed by refinement. The 2F_o − F_c electron density map is contoured at lσ (blue), the F_o − F_c map is contoured at 3σ (green) and −3σ (red). d The SiaT–Neu5Ac interaction network represented as a Ligplot⁺ diagram. Hydrogen bonds (dashed lines), hydrophobic contacts (arcs with spokes) and interacting water molecules (yellow) are shown

Fig. 2

Characterisation of SiaT. a SiaT is able to rescue growth of E. coli ∆NanT on Neu5Ac as the sole carbon source. The growth lag observed for ΔNanT+pNanT and ΔNanT+pSiaT is due to IPTG induction of the T5 promoter on pNanT and pSiaT. Growth curves represent the mean of six experiments ± SEM. b Time course of Neu5Ac uptake into proteoliposomes reconstituted with SiaT. In (black circle, black square, white triangle, black triangle), valinomycin was added to facilitate K⁺ movement prior to transport. In (white circle), ethanol was added instead of valinomycin as a control. In (white circle, black square, black circle), 10 mM NaCl was added together with [³H]-Neu5Ac; in (white triangle) 10 mM KCl was used in place of NaCl; in (black triangle) no salts were used in the transport assay. In (black square), transport was measured in empty liposomes. On the left Y-axis, specific transport activity is reported; on the right Y-axis transport in empty liposomes. is reported. Uptake data were fitted in a first-order rate equation for time course plots. c The transport of [³H]-Neu5Ac in the presence of 10 mM NaCl was measured in proteoliposomes reconstituted with SiaT, with an imposed K⁺ diffusion membrane potential. Data were plotted using the Michaelis–Menten equation. d The kinetics of Neu5Ac transport by SiaT sialic acid binding site variants. The transport of [³H]-Neu5Ac with or without NaCl was measured in proteoliposomes reconstituted with wild type and mutated variants, with an imposed K⁺ diffusion membrane potential. All proteoliposome measurements (b–d) are presented as means ± SD from five independent experiments. e MST binding assay of Neu5Ac binding to SiaT. f Representative isothermal titration calorimetry raw data (top) and binding isotherm (bottom) of Neu5Ac binding with SiaT. g Chemical structures of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and ketodeoxynononic acid (KDN). h, i MST binding assay of Neu5Gc (h) and KDN (i) binding to SiaT. j Determination of the SiaT Na⁺ Hill coefficient. Data were plotted using the Hill equation. The inset represents the same data plotted using a log-scale for the X-axis to increase the resolution of low concentration data points. MST (e, h, i) and ITC (f) experiments represent the mean of three independent experiments ± SEM; for each, data from one representative experiment is shown

Fig. 3

Amino-acid sequence alignment and secondary structure of P. mirabilis SiaT with SiaT transporters from eight additional species of bacteria. SiaT transporters from Morganella morganii, S. enterica, Vibrio fischeri, Plesiomonas shigelloides, Photobacterium profundum, S. aureus, C. perfringens, Clostridium difficile and S. pneumoniae are aligned. Residues are numbered according to P. mirabilis SiaT, and the corresponding secondary structure of this transporter is shown above the alignment, with α-helices depicted as coils. Residues highlighted with black boxes are conserved, residues implicated in sialic acid binding are highlighted below with an orange asterisk and residues involved in sodium-binding are highlighted below with a blue asterisk

Fig. 4

The sodium-binding sites. a Amino-acid residues (sticks coloured by atom type) coordinating the two Na⁺ ions (blue spheres). Inset depicts the omit map for the Na⁺ ions generated by removing respective ligands from the X-ray structure followed by refinement. The 2F_o − F_c electron density map is contoured at lσ (blue), the F_o − F_c map is contoured at 3σ (green). b Ligplot⁺ analysis of the SiaT and Na⁺ ion interactions. Na⁺ ion coordination is indicated by dashed lines between the atoms involved. c The kinetics of Neu5Ac transport by SiaT sodium-binding site variants. The transport of [³H]-Neu5Ac with or without NaCl was measured in proteoliposomes reconstituted with wild-type SiaT and mutated variants (Ser342Ala, Ser343Ala, Ser345Ala, Ser346Ala and Asp182Ala) with an imposed K⁺ diffusion membrane potential. Ser342Ala and Ser343Ala correspond to the Na2 site, whereas Ser345Ala, Ser346Ala and Asp182Ala correspond to the Na3 site. All proteoliposome measurements are presented as means ± SD from five independent experiments

Fig. 5

Sodium and substrate binding sites of Na⁺ transporters that adopt the LeuT fold. a SiaT (outward-open, pdbid: 5NV9), b vSGLT (inward-open, pdbid: 3DH4), c Mhp1 (outward-occluded, pdbid: 4D1B), d BetP (Asp153Gly outward-open, pdbid: 4LLH), e LeuT (outward-occluded, pdbid: 2A65), f dDAT (N-terminally truncated, EL2 deleted, Val74Aala, Val275Ala, Val311Ala, Leu415Ala, Gly538Leu, pdbid: 4M48) and f SERT (outward-open, Tyr110Ala, Ile291Ala, Thr439Ser, Cys554Ala, Cys580Ala, Cys622Ala, pdbid: 5I71). Substrates and amino-acid residues coordinating the Na⁺ ions are represented in sticks with carbon atoms in grey or white, respectively. Residues surrounding the putative Na1´ in BetP are shown in sticks (d). Oxygen atoms are red and nitrogen atoms are blue. The Na⁺ ions are represented as blue spheres

Fig. 6

Molecular dynamics simulations of SiaT. a Heavy-atom RMSD of the substrate with respect to the X-ray structure for four MD simulations with different combinations of Na⁺ ions in the Na2 and Na3 sites (see legend, upper right). Snapshots compare the instantaneous configuration of Neu5Ac, Arg135, Na2 (when present), and unwound residues in Tm1 (Thr58, Leu59 and Ser60) with the X-ray structure (ghost). Solid lines are smoothed over an 800 ps window, while the original full data set saved every 40 ps is transparent. b–e Dependence of Leu59 backbone motion on Na⁺ ion occupancy. ϕ and ψ angles for Leu59 from all four simulations with Neu5Ac bound and different combinations of Na⁺ ions in the Na2 and Na3 sites. Both Na2 and Na3 bound (b), Na2 bound only (c), Na3 bound only (d) and no ions bound (e). In each panel, the instantaneous angle pairs are plotted every 40 ps over the entire 200 ns simulation (light blue), and the data set is contoured at values of 100 (black), 50 (red) and 10 (yellow). The ϕ and ψ values in the X-ray structure are represented as a black dot. The distributions in panels d and e, which lack an ion in the Na2 site, are so broad that the black high-density contour does not exist. f, g Ion stability in the Na2 and Na3 sites. Simultaneous distance of bound Na⁺ ions to the Na2 site and the Na3 site from 200 ns MD simulations in the presence or absence of Neu5Ac with both Na⁺ ions bound (f), only the Na2 ion bound (g) and only the Na3 ion bound (h). In all panels, every point represents a simulation frame saved every 40 ps, the Na2 ion position is blue (with substrate) or green (without substrate), and the Na3 ion position is red (with Neu5Ac) or yellow (without Neu5Ac)

Fig. 7

The outward open structure and an inward open model of SiaT. a, b Surface representation of the outward open structure (a) and the inward open model (b). Neu5Ac is shown in grey sticks coloured by atom type. Positive potential is shown in blue and negative potential in red. c, d Predicted movement of transmembrane helices between the outward open structure (coloured) and the inward open model (white) at the periplasmic side. Transmembrane helices implicated in the conformational change between states are labelled. Neu5Ac is shown in grey sticks coloured by atom type and the Na⁺ ions are depicted as blue spheres. Hydrophobic gate residues are depicted as spheres coloured by atom type (d). e Movement of transmembrane helices between the outward open structure (coloured) and the inward open model (white) at the cytoplasmic side. The cap helix (Ilh0), transmembrane helices and amino acids implicated in opening and stabilising the inner gate are highlighted. f Transmembrane helices and amino-acid interactions that stabilise the opening of the gate at the cytoplasmic side are highlighted