Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins

Abstract

The principal virulence factor of human pathogenic enterohemorrhagic Escherichia coli is Shiga toxin (Stx). Shiga toxin 2a (Stx2a) is the subtype most commonly associated with severe disease outcomes such as hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 subunit (Stx2A1) binds to the conserved elongation factor binding C-terminal domain (CTD) of ribosomal P stalk proteins to inhibit translation. Stx2a holotoxin also binds to the CTD of P stalk proteins because the ribosome-binding site is exposed. We show here that Stx2a binds to an 11-mer peptide (P11) mimicking the CTD of P stalk proteins with low micromolar affinity. We cocrystallized Stx2a with P11 and defined their interactions by X-ray crystallography. We found that the last six residues of P11 inserted into a shallow pocket on Stx2A1 and interacted with Arg-172, Arg-176, and Arg-179, which were previously shown to be critical for binding of Stx2A1 to the ribosome. Stx2a formed a distinct P11-binding mode within a different surface pocket relative to ricin toxin A subunit and trichosanthin, suggesting different ribosome recognition mechanisms for each ribosome inactivating protein (RIP). The binding mode of Stx2a to P11 is also conserved among the different Stx subtypes. Furthermore, the P stalk protein CTD is flexible and adopts distinct orientations and interaction modes depending on the structural differences between the RIPs. Structural characterization of the Stx2a-ribosome complex is important for understanding the role of the stalk in toxin recruitment to the sarcin/ricin loop and may provide a new target for inhibitor discovery.

Keywords: E. coli; Escherichia coli; P1/P2 protein; RIP; Shiga toxin; bacterial toxin; ribosomal stalk; ribosome; ribosome inactivating protein; toxin; translation.

Figure 1.

Model depicting the recruitment of Stx2A1 by P stalk to gain access to the ribosomal SRL. The schematic representation of P0 fragment linked with the N-terminal domain of the P proteins (green) (PDB ID: 3A1Y) from Archaea superpositioned onto the S. cerevisiae 60S subunit. The S. cerevisiae 26S rRNA (PDB ID: 3U5H) and 60S subunit (PDB ID: 3U5I) are indicated as dark gray and light gray sticks, respectively. One CTD of a P protein is shown as a gray line attached to the Stx2A1 molecule with a gray circle at the end. The yeast 60S subunit is oriented to show the SRL (red) along with Stx2A1 positioned to show the putative SRL binding site (cyan) and the P stalk–binding pocket (orange) on Stx2A1.

Figure 2.

Interaction of P11 with Stx2a. Stx2a was immobilized on Fc2 of Biacore T200 at 4364 relative units. Fc1 was activated and blocked as reference. P11 was passed over the surface at six different concentrations (5–1215 μm). The association and dissociation times were for 30 s at a flow rate of 50 μl/min. The running buffer was PBS-P with 2% DMSO. The data were solvent corrected and fit with Biacore T200 software 3.0. The analysis was repeated three times.

Figure 3.

Structure of Stx2a-P11 complex. A, structure of Stx2a with Stx2A (green) and the Stx2B pentamer (yellow, magenta, cyan, salmon, and light gray) depicted as a ribbon diagram in complex with the P11 peptide (blue) drawn as sticks. Stx2A1 active site residue Tyr-77 is drawn as sticks and colored red. B, original 2F_o − F_c (blue mesh) and F_o − F_c (red mesh) electron density maps of the P11 peptide drawn as sticks at the binding pocket of Stx2a with carbon and nitrogen atoms blue and oxygen atoms red. The 2 F_o − F_c electron density map is presented at 1.0 σ level, whereas the F_o − F_c electron density map is presented at 3.0 σ level. The maps were calculated before P11 was built into the density maps. The P11 primary sequence depicted in bold text. C, close-up of the Stx2A1 active site occluded by Stx2A2. Stx2A1 (green) active site residue Tyr-77 is drawn as sticks and colored red. Stx2A2 residue Glu-259 is color coordinated to the main chain color with oxygen atoms red. The proximity of Tyr-77 and Glu-259 is highlighted by blue dashes.

Figure 4.

Stx2A P11-binding mode. A, semitransparent electrostatic surface potential map of the P11 (blue sticks) binding pocket on Stx2A. The surface color represents electric potential with red color as negatively charged surface, blue color as positively charged surface, and neutral regions colored in white. Neighboring residues to the P11 peptide are drawn as sticks with carbon atoms gray, nitrogen atoms blue, and oxygen atoms red. B–D, close-up of the key interactions between Stx2A (green ribbon) in complex with P11 (blue sticks) depicting the (B) salt bridges, (C) H-bonds, and (D) nonpolar interactions between Stx2A and P11. All side chains are drawn as sticks and color coordinated to the main chain color with nitrogen atoms blue and oxygen atoms red. Salt bridges are represented as red dashes in (B) and hydrogen bonds are represented as yellow dashes in (C).

Figure 5.

Different P11-binding modes of Stx2A, RTA, and TCS. A–E, molecular surface representation of Stx2A (gray surface) with P11 peptide (blue) with the superpositioned P11 peptides from RTA (light teal) and TCS (dark salmon) depicting different P11-binding modes of all three proteins. All P11 peptides are drawn as Cα-traces. Electrostatic surface potential map of the P11-binding pocket on (B) RTA and (D) TCS. The semitransparent surface color represents electric potential with red color as negatively charged surface, blue color as positively charged surface, and neutral regions colored in white. P11 is drawn as sticks and colored light teal bound to RTA and dark salmon bound to TCS. Residues adjacent to the P11 in panels B and D are drawn as sticks with carbon atoms light teal for (B) RTA and dark salmon for (D) TCS with nitrogen atoms blue, and oxygen atoms red. Close-up of the key interactions between (C) RTA (gray ribbon) with P11 drawn as sticks with carbon atoms colored light teal, oxygen colored red, and nitrogen colored blue and (E) TCS (gray ribbon) in complex with P11 drawn as sticks with carbon atoms colored dark salmon, oxygen colored red, and nitrogen colored blue. Salt bridges and hydrogen bonds are represented as red dashes in (C and E). RTA and TCS side chains are drawn as sticks and color coordinated to the main chain color with nitrogen atoms blue and oxygen atoms red.