Molecular basis of differential B-pentamer stability of Shiga toxins 1 and 2

Abstract

Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx), an AB(5) toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown, data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC(50) = 2.3 µM vs. EC(50) = 0.043 µM for Stx1B). X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1) within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.

Figure 1. Differential solution stability of Stx1B and Stx2B pentamers.

A. Sedimentation coefficient distribution plots from analytical ultracentrifugation sedimentation velocity experiments show a major peak at ∼3 S, which Sedfit estimates to have a molecular weight consistent with pentamer, at 0.8 to 2 µM loading concentrations. B. The sedimentation coefficient distributions of Stx2B at concentrations ranging from 0.5 to 8 µM demonstrate significant differences in solution state assembly. Stx2B appears to be stably pentameric at 4 to 8 µM, decreasing to mostly monomeric at 0.5 µM. C and D. Representative plots of sedimentation equilibrium data from global fits of three concentrations and four speeds in WinNonlin confirm a monomer-pentamer equilibrium for Stx1B and Stx2B. The K₅ and EC₅₀ values determined from these fits are listed in Table 3.

Figure 2. Prediction of Gln40 as a destabilizing amino acid in Stx2B.

A. Alignment of Stx1B and Stx2B sequences, with identical residues identified with a dash. Residues L41 and Q40 are marked. B. Structure of the Stx2 B-pentamer (PDB ID: 1R4P) indicating Gln40 (cyan) and Leu38 (blue). The polar side chain of Gln40 occupies an otherwise hydrophobic pocket at the interface between α-helices of adjacent B-subunits. Inset is a zoomed view of Leu38 and Gln40 from adjacent monomers in Stx2B. The close proximity of Leu38 and Gln40 (<5 Å) was predicted to disrupt monomer packing. In contrast, a leucine occupies position 41 in Stx1B, which may help stabilize the hydrophobic interface between B-subunits.

Figure 3. Sedimentation velocity of reciprocal point mutants.

Sedimentation velocity c(s) traces for Stx1B-L41Q (A) and Stx2B-Q40L (B) demonstrate a reversal in the pentamer stability in the reciprocal mutants compared to wild-type. Stx1B-L41Q disassembles over the same concentration range that Stx1B wt retains stable pentamer assembly, and Stx2B-Q40L is stabilized relative to wt Stx2B.

Figure 4. Circular dichroism spectra of wild-type and mutant proteins.

A. Stx1B wild-type and L41Q mutant CD wavelength spectra. The far-UV spectra overlay, suggesting that no large-scale changes in secondary structure occur in the L41Q point mutant. B. Stx2B wild-type and Q40L mutant CD spectra. The spectra overlay, supporting the structural similarity between wild-type and Q40L mutant proteins.

Figure 5. Representative species concentration plots of monomeric and pentameric B-subunits from sedimentation equilibrium data.

Stx1B (A) and Stx2B-Q40L (D) show predominantly pentamer species. Stx2B (B) is primarily monomeric, whereas Stx1B-L41Q (C) retains a mixture of monomer and pentamer species. K₅ and EC₅₀ values for these data are listed in Table 3.

Figure 6. Thermal denaturation of Stx isoforms and mutants.

A. Stx1B (black) and Stx1B-L41Q (gray) thermal denaturation curves. Stx1B-L41Q is destabilized (decreased melting temperature) compared to wild-type Stx1B. B. Stx2B (black) and Stx2B-Q40L (gray) thermal denaturation curves show that Stx2B-Q40L has an increased thermal stability relative to wild-type Stx2B. Parameters from the fits can be found in Table 4.

Figure 7. Superimposed crystal structures of Stx2B and Stx2B-Q40L subunits.

Alpha-carbon traces from a representative B-subunit from the published structure of wild-type Stx2 (gray) (PDB ID: 1R4P) and Stx2B-Q40L (green) overlay. The backbone RMSD of 0.260 to 0.555 Å indicates no major structural rearrangements occurred in the Stx2B-Q40L mutant.

Figure 8. Side-chain packing for Stx2B and Stx2B-Q40L.

A. A cartoon model of a single wild-type Stx2B subunit with a space-filling model of the Q40 side chain is shown in gray, and the surface of the adjacent B-subunit is colored by charge (blue  =  positive, red  =  negative, white  =  neutral). The oxygen and nitrogen atoms (colored red and blue, respectively) of the Q40 side chain lie in an unfavorable position in the inter-subunit interface. B. By contrast, the Stx2B-Q40L mutant (in green) shows the L40 side chain tightly packed into the hydrophobic pocket.