Furanose-specific sugar transport: characterization of a bacterial galactofuranose-binding protein

Abstract

The widespread utilization of sugars by microbes is reflected in the diversity and multiplicity of cellular transporters used to acquire these compounds from the environment. The model bacterium Escherichia coli has numerous transporters that allow it to take up hexoses and pentoses, which recognize the more abundant pyranose forms of these sugars. Here we report the biochemical and structural characterization of a transporter protein YtfQ from E. coli that forms part of an uncharacterized ABC transporter system. Remarkably the crystal structure of this protein, solved to 1.2 A using x-ray crystallography, revealed that YtfQ binds a single molecule of galactofuranose in its ligand binding pocket. Selective binding of galactofuranose over galactopyranose was also observed using NMR methods that determined the form of the sugar released from the protein. The pattern of expression of the ytfQRTyjfF operon encoding this transporter mirrors that of the high affinity galactopyranose transporter of E. coli, suggesting that this bacterium has evolved complementary transporters that enable it to use all the available galactose present during carbon limiting conditions.

FIGURE 1.

Characterization of ligand binding to YtfQ. a, ES-MS spectrum of 5 μm YtfQ under denaturing conditions. The molecular mass of the unbound protein (U) is indicated at 33,190 atomic mass units (amu). Possible phosphate adducts, of 98 amu are indicated by asterisks. Inset, Coomassie-stained SDS-PAGE gel containing 2.4 μg of purified YtfQ (lane 1) and molecular weight markers (lane 2). b, ES-MS spectrum of 10 μm YtfQ under native conditions in 50 mm ammonium acetate pH5. The unbound protein (U) is the primary peak at the indicated mass of 33,189 with an additional ligand-bound species (L) at 33,369 amu. Acetate adducts are indicated with hashes. c, galactose binding to 0.19 μm YtfQ in 50 mm Tris-HCl, pH 8, as measured by tryptophan fluorescence spectroscopy.

FIGURE 2.

Crystal structure of YtfQ. a, schematic of the structure of YtfQ colored with rainbow coloring from the N terminus (blue) to the C terminus (yellow). The galactofuranose is shown in a stick representation. The single disulfide bond joining residues Cys-129 and Cys-193 is indicated in dark blue. b, topology diagram of YtfQ. The N-terminal domain is in white and the C-terminal domain in gray. Circles represent α-helices, triangles represent β-strands, and unfilled ovals are 3₁₀ helices. The double line from β6 indicates the position of the disulfide bond. c, structure of galactofuranose modeled into electron density contoured at 1.5 σ.

FIGURE 3.

Stereo image of the binding site of YtfQ containing both α- and β-galactofuranose. The amino acids involved in coordinating the ligand are represented in thin stick format and are labeled, and the ligand is represented in thick stick format. Hydrogen bonds are indicated with dashed lines.

FIGURE 4.

Superposition of the ligand binding sites of YtfQ and RbsB. The amino acid side chains involved in coordinating ribopyranose by RbsB (cyan) or galactofuranose by YtfQ (green) are labeled by their positions in the PDB files 2DRI and 2VK2, respectively. The coordination of galactofuranose also involves a water (red sphere).

FIGURE 5.

Multiple structure-based alignment of the amino acid sequences of E. coli YtfQ, AlsB, RbsB, and Salmonella typhimurium MglB proteins. Triangles indicate residues in contact with ligand in YtfQ (red direct, blue indirect). Blue stars indicate the aromatic residues that form part of the binding pocket. The cysteine residues (Cys-129 & Cys-193) that form the disulfide bond are labeled (green numeral 1).

FIGURE 6.

NMR analysis of ligands present in YtfQ. a, one-dimensional ¹³C projections of three ¹H-¹³C HSQC experiments. The upper panel shows the [¹³C₁]galactose after denaturation of YtfQ by addition of d₆-DMSO. The middle panel shows the projection of the HSQC acquired 4 days later. The lower panel shows the projection of the HSQC acquired of the lyophilized dialysis buffer after re-dissolving in d₆-DMSO. b, volume of the peaks in the corresponding two-dimensional spectra (supplemental Fig. S4). Peak assignments are according to those published previously (30). Peaks at the ¹³C chemical shifts of β-galactofuranose (β-f), α-galactofuranose (α-f), β-galactopyranose (β-p), and α-galactopyranose (α-p) are indicated. The large peak at ∼100.5 ppm arises from folding of the DMSO resonance.