Structural basis of subtilase cytotoxin SubAB assembly

Abstract

Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhance or inhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.

Keywords: AB5 Toxins; Carbohydrate-binding Protein; Cellular Trafficking; Disassembly/Assembly; Infectious Diseases; Intracellular Trafficking; Structural Biology; Toxins.

FIGURE 1.

Overall architecture of SubAB. The A1 component of the A-subunit (SubA) is shown in light blue, whereas the α-helix A2 is shown in blue. The disulfide tethering the A1 and A2 is shown in red. Each protomer of the pentameric B-subunit (SubB) is colored in cyan, yellow, lemon, salmon, and pale green. The figure was generated using PyMOL (29).

FIGURE 2.

A, overall superposition of the isolated crystal structure of SubA and the SubAB holotoxin. The crystal structure of SubA (PDB code: 2IY9) is colored in gray, and the A-subunit in the SubAB structure is colored in salmon. The B-subunit of SubAB is shown in light blue. B, structural rearrangement of the loop (Ser-135–Pro-141). C, structural rearrangement of the loop (Asp-41–Pro-45). D, structural rearrangement of the residue Arg-291 upon formation of the holotoxin. The figures were generated using PyMOL (29).

FIGURE 3.

Hydrogen bond network between the SubA and SubB residues. The hydrogen bonds are shown as red dashed lines.

FIGURE 4.

A, The molecular surfaces of residues Tyr-66, Thr-69, Thr-70, and Gly-71 are colored by protomer, and the positions of those residues are also shown as sticks. B, calculated molecular electrostatic surface potential of three SubB protomers shown in two orientations. The Tyr-66, Thr-69, Thr-70, and Gly-71 surface locations are indicated. Negative surfaces are in red (−10 kT), neutral surfaces are in white (0 kiloteslas), and positive surfaces are in blue (10 kiloteslas). The surface electrostatic potential map was calculated using the nonlinear mode of the Adaptive Poisson Boltzmann Solver (APBS) (30, 31). The figures were generated using PyMOL (29).

FIGURE 5.

A, structure-based sequence alignment of the B-subunit α1-helix of members of the four AB₅ toxin families. The residues that form the hydrophobic patch are boxed in orange. The gray shadowing indicates the sequence identity level between the six sequences. The SubB secondary structural elements are shown. The figure was generated using the Indonesia program (16). B, calculated molecular electrostatic surface potential of members of the four AB₅ toxin families (SubAB, LT-I, LT-IIb, Ctx, Stx, and Stx2). For clarity, the surface of three protomers for each AB₅ toxins (1 to 3) is shown. The molecular electrostatic surface potential was calculated as described in the legend for Fig. 4. The figure was generated using PyMOL (29).

FIGURE 6.

Positions of the 13 mutated SubB residues. The locations of the residues belonging to protomer C are shown. Segments of the A1 domain and the A2 helix (SubA) are shown and colored in blue and yellow, respectively. For clarity, only the α-helix of the other four protomers is shown (in green). The figure was generated using PyMOL (29).

FIGURE 7.

Effect of SubB mutations on cytotoxic activity of SubAB. A, SubAB cytotoxicity was assayed on Vero cell monolayers as described under “Experimental Procedures.” Data are expressed as a percentage of wild type activity. Data are representative of two independent experiments. B, relative dissociation of SubA bound to SubB and SubB mutants after incubation in detergent (30 mm 3-(decyldimethylammonio)propanesulfonate inner salt). SubA quantities were analyzed by SDS-PAGE and densitometry. SubA quantities for each SubB mutant were normalized to the wild type SubA dissociation. Error bars indicate mean ± S.E. of triplicate samples.

FIGURE 8.

Intracellular trafficking of SubAB mutants. Vero cells were treated with 3 μg ml⁻¹ purified wild type SubAB or the indicated mutant derivatives for 30 min, and then fixed, permeabilized, and differentially stained for SubA (red) or SubB (green) using immunofluorescence, as described under “Experimental Procedures.” A, separate and overlaid confocal images are shown for SubA and SubB; in the overlay, nuclei are stained with DAPI (blue). B, SubB/SubA fluorescence intensity ratios were calculated either on whole fields or on the ER compartment. Data are the mean ± S.D. ratios for at least six fields/cells (*, p < 0.0001; **, p = 0.0001; unpaired Student's t test (relative to wild type ratio)).