Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding

Abstract

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates.

Keywords: bacterial pathogenesis; membrane transporter reconstitution; protein structure; sialic acid; transporter.

FIGURE 1.

Neu5Ac is bound by SiaP-His₆ with high affinity. A, tyrosine fluorescence changes of 0.05 μm SiaP-His₆ upon increasing additions of Neu5Ac. B, fluorescence emission spectra of 0.05 μm SiaP-His₆:F170W before and after the addition of 20 μm indicating the large quench in signal due to binding. A buffer only control is included to illustrate the scattering light from the emission signal. C, isothermal titration calorimetry analysis of 10 μm SiaP-His₆ in 50 mm Tris/HCl, pH 8, at 37 °C. 150 μm Neu5Ac was injected into the cell in 6-μl aliquots.

FIGURE 2.

Effects of the Arg-147 mutants on Neu5Ac transport via SiaPQM are due to reduced affinity for the ligand. A, in vivo uptake of 5 μm [¹⁴C]Neu5Ac by E. coli BW25113 ΔnanAT mediated by IPTG-induced expression of siaPQM and variants from pWKS30. Inset shows the data for the Arg-147 mutant expanded to illustrate the detectable activity of the R147K mutant (diamonds). B, in vitro uptake of 5 μm [¹⁴C]Neu5Ac into SiaQM-containing proteoliposomes with a pre-generated ΔNa⁺ Δψ across the membrane mediated by 5 μm SiaP-His₆ and variants. The negative control included used liposomes lacking SiaQM. C, growth of E. coli BW25113 ΔnanT in M9 minimal medium with 3.2 mm (1 mg/ml) Neu5Ac as the sole carbon source mediated by IPTG-induced expression of siaP-His₆–siaQM and variants. Growth is reported as an apparent A₆₅₀ (A₆₅₀*) due to the data output of the experimental system. Positive and negative controls are shown as filled and empty circles, respectively, and the R147A, R147K, and R147E mutants are represented by empty down triangles, diamonds, and up triangles, respectively. All data are an average of three or four repeat experiments. D, final A₆₅₀ values of overnight cultures supplemented with 0.4% glucose (black), 3.2 mm Neu5Ac (pale gray), and 3.2 mm KDN (dark gray) as the sole carbon sources.

FIGURE 3.

Ligand binding induces closure of the mutants with waters substituting missing direct interactions. A, structural superposition of mutants R147K (magenta) and R147A (green) onto WT (gray, shown with side chains) highlights that all structures exhibit a closed conformation in the presence of Neu5Ac (blue) and are structurally indistinguishable. B, close-up of the binding pocket of the R147K mutant solved to 2.2 Å resolution, with electron density (2mF_o − DF_c omit maps rendered at 1σ) shown, reveals that two water molecules 1 and 2 (red spheres labeled with blue font) take up interactions with opposite ends of the ligand enabled by the alternative conformation of Asn-10 and the Lys mutation. C, close-up of the binding pocket of R147A (2mF_o − DF_c omit maps rendered at 1.5σ). The Ala mutation opens space for another water (3) in an equivalent position to the missing guanidinium group, but without altering the surrounding backbone in comparison with the wild type (shown in gray lines).

FIGURE 4.

Growth of E. coli strains on 1 mm Neu5Ac as the sole carbon source. Growth of E. coli strains in M9 minimal medium with 1 mm Neu5Ac as the sole carbon source over 48 h with the indicated strains: wild-type (WT) (gray diamond); ΔnanT strain (gray circle); this same strain complemented the wild-type siaPQM (dark gray triangles) or the genes with a siaP mutant allele of R147A (white triangles), R147E (black squares), or R147K (crosses). Data are the average of at least triplicate experimental replicates.

FIGURE 5.

Molecular fishing for organic acids. A, both WT and R147E mutant have been exposed to a stock of non-cognate “sialylamide.” In the absence of apo-structures, 2CEY was used to depict the open form of the structure, colored from blue to red from the N to the C terminus. B, R147E (cyan sticks) exhibits a closed conformation in the presence of sialylamide, superposed onto WT (gray lines for side chains and stars for water molecules) for reference. Two waters (red spheres, 1 and 3) bridge the interaction between the amide and the mutated residue R147E (red dashed lines). Electron density (2mF_o − DF_c omit maps rendered at 3σ) is shown for several other waters (5–7) that are shifted in response to the amide H-bond donor. C, however, atomic resolution (2mF_o − DF_c omit maps rendered at 5σ) reveals that the wild-type protein filters out a minor impurity of sialic acid confirming that the conserved arginine confers high selectivity for organic acids; cf. same position of surrounding water molecules as in WT in gray.