Structural Basis for High Specificity of Amadori Compound and Mannopine Opine Binding in Bacterial Pathogens

Abstract

Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. Opines are either sugar phosphodiesters or the products of condensed amino acids with ketoacids or sugars. They are Agrobacterium nutrients and imported into the bacterial cell via periplasmic-binding proteins (PBPs) and ABC-transporters. Mannopine, an opine from the mannityl-opine family, is synthesized from an intermediate named deoxy-fructosyl-glutamine (DFG), which is also an opine and abundant Amadori compound (a name used for any derivative of aminodeoxysugars) present in decaying plant materials. The PBP MotA is responsible for mannopine import in mannopine-assimilating agrobacteria. In the nopaline-opine type agrobacteria strain, SocA protein was proposed as a putative mannopine binding PBP, and AttC protein was annotated as a mannopine binding-like PBP. Structural data on mannityl-opine-PBP complexes is currently lacking. By combining affinity data with analysis of seven x-ray structures at high resolution, we investigated the molecular basis of MotA, SocA, and AttC interactions with mannopine and its DFG precursor. Our work demonstrates that AttC is not a mannopine-binding protein and reveals a specific binding pocket for DFG in SocA with an affinity in nanomolar range. Hence, mannopine would not be imported into nopaline-type agrobacteria strains. In contrast, MotA binds both mannopine and DFG. We thus defined one mannopine and two DFG binding signatures. Unlike mannopine-PBPs, selective DFG-PBPs are present in a wide diversity of bacteria, including Actinobacteria, α-,β-, and γ-proteobacteria, revealing a common role of this Amadori compound in pathogenic, symbiotic, and opportunistic bacteria.

Keywords: ABC transporter; DFG; crystal structure; host-pathogen interaction; isothermal titration calorimetry (ITC); mannopine; opine; periplasmic binding protein; plant pathogen; x-ray crystallography.

FIGURE 1.

Metabolism of DFG (orange) and mannopine (green) in transformed plant cells and the two A. tumefaciens B6 (up) and C58 (down) strains. In plant tumor cells the genes mas2 (dark red) and mas1 (blue) responsible for the biosynthesis of DFG and mannopine, respectively, are located on the T-DNA transferred from the A. tumefaciens B6 Ti plasmid. In A. tumefaciens B6, the genes (motA-D) coding for mannopine import on the Ti plasmid are shown in purple. Mannopine is converted into DFG, which in turn is deconjugated into glutamine and fructose by the reverse synthesis reactions. No DFG transporter has been identified yet. In A. tumefaciens C58 strain, mannopine and DFG were proposed to be both transported by the products of socAB (slate) located on the At plasmid. DFG is degraded by the products of socCD into glutamine and fructose (40). The AttC (gene in light brown) was annotated as a mannopine binding-like protein.

FIGURE 2.

ITC and fluorescence K_D measurements. The top graphs of MotA (A) and SocA (B) microcalorimetry experiments show heat differences upon injection of ligand (top panel) and integrated heats of injection with the best fit (solid line) to a single binding model using Microcal ORIGIN (low panel). The lower graphs show fluorescence monitoring upon titration with each ligand and fit (solid line) to a single binding model using Origin 7. Measures were done in triplicate. C, calculated parameters for each experiment are indicated.

FIGURE 3.

Ribbon representation of MotA structures with mannopine in green (A) and unliganded (B). Lobes I and II are shown in deep purple and pink, respectively, and the hinge region is in red. C, comparison between the open unliganded (deep purple) and the closed liganded (green) forms of MotA. D, packing of the unliganded MotA crystal; the N terminus His₆ tag (shown in sticks) of a molecule (deep purple) of the asymmetric unit enters the ligand binding site of its symmetric molecule (green).

FIGURE 4.

Mannopine (A), DFG (B), and glucopine (C) bound to the binding site of MotA are shown in gray/green, gray/orange, and gray/deep blue sticks, respectively, and in their annealing F_o − F_c omit map contoured at 4σ. Hydrogen bonds between MotA and each ligand are shown as dashed lines in black (distance below 3.2 Å) and in green (distance between 3.2 and 3.4 Å). D, superposition of the three ligands in the binding site of MotA. The residues in green interact with mannopine, in orange with DFG, and in deep blue with glucopine.

FIGURE 5.

DFG (A) and glucopine (B) bound to the binding site of SocA are shown in gray/orange and gray/deep blue sticks, respectively, and in their annealing F_o − F_c omit map contoured at 4σ. Hydrogen bonds between SocA and each ligand are shown as dashed lines in black (distance below 3.2 Å) and in green (distance between 3.2 and 3.4 Å). C, superposition of both ligands in the SocA binding site. The residues in orange interact with DFG and in deep blue with glucopine.

FIGURE 6.

MotA phylogeny and mannopine binding signature. The displayed tree was rooted with AttC sequence. For each protein or protein cluster, the residues which are identical to (black) and different from (red) those involved in the binding of the glutamine part (purple box) and sugar part (green box) of mannopine are indicated.

FIGURE 7.

SocA phylogeny and DFG binding signature. The branches are collapses when the clade is composed of proteins from a same genus and are colored in red for α-Proteobacteria, purple for β-Proteobacteria, blue for γ-Proteobacteria and green for Actinobacteria. For each protein or protein cluster, the residues which are identical to (black) and different from (red) those involved in the binding of DFG via their main chain (orange box) and via their side chain (purple box) are indicated.