Charged and hydrophobic surfaces on the a chain of shiga-like toxin 1 recognize the C-terminal domain of ribosomal stalk proteins

Abstract

Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A(1) chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A(1) chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A(1) chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A(1) variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A(1) chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A(1) chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A(1) chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs.

Figure 1. The A₁ chain of SLT-1 binds to a conserved C-terminal ribosomal peptide.

(A) Amino acid sequence representing the 17-residue C-terminus common to ribosomal stalk proteins P1 and P2. The last 11 amino acids (underlined) delimit the shortest peptide element shown to interact with the A₁ chain . (B) Relative surface plasmon resonance (SPR) signals for the A₁ chain of SLT-1 binding to immobilized, biotinylated monomeric synthetic peptides were plotted as a function of SLT-1 A₁ chain concentration. The calculated dissociation constants (K_d) suggest that both monomeric peptides have similar affinities for the A₁ chain. Each point on the curve represents the average relative SPR signals from experiments performed in quadruplicate.

Figure 2. Surface plasmon resonance analysis of alanine-containing peptide variants of the conserved C-terminal ribosomal stalk peptide SDDDMGFGLFD confirms that the interaction with the A₁ chain of SLT-1 requires both electrostatic and hydrophobic contacts.

The peptide sequence corresponding to the final 11 residues of the conserved C-terminal peptide (SDDDMGFGLFD) was substituted at each position for an alanine residue. Individual peptides corresponding to a substitution of charged (Panel A) or other residues (Panel B) were biotinylated and immobilized on an NLC SPR sensor chip. Each monomeric peptide was exposed to ten 2-fold serial dilutions of the A₁ chain of SLT-1 in triplicate and the responses were subtracted from buffer alone and a control peptide. The SPR responses for the single and double/triple alanine variants were graphed and compared to the control natural peptide. Amino acid substitutions that resulted in a peptide that lacked an interaction with the A₁ chain of SLT-1 could not be plotted. Calculated dissociation constants are reported in Table 1.

Figure 3. The A₁ chain of SLT-1 harbors a cationic surface composed of a cluster of arginine residues that interact with the ribosomal stalk protein P2 and the conserved C-terminal peptide.

(A) A vector expressing a catalytically inactive variant of the SLT-1 A₁ domain (CIA₁) or one of the arginine-to-alanine point mutants as fusion partners with the GAL4 DNA-BD domain were co-transformed in the yeast strain AH109 with a vector expressing ribosomal protein P2 as a fusion construct to the GAL4-AD. The transformed yeast cells were plated on SD agar −Trp/−Leu. The resulting yeast colonies were grown overnight, and spotted (10 µl) as 10-fold serial dilutions onto SD medium lacking Trp and Leu to select for the presence of each plasmid followed by spotting on SD media lacking Trp, Leu, and His to select for interacting partners leading to colony growth. (B) SPR profiles illustrating the decrease in relative units for the arginine-to-alanine SLT-1 A₁ chain variants in relation to the wild-type A₁ chain, at a concentration of 15 µM, when presented to the immobilized peptide SDDDMGFGLFD. (C) Increasing salt concentrations led to a decrease or loss of binding of wild-type SLT-1 A₁ chain when exposed to the peptide SDDDMGFGLFD. SPR traces were plotted for the wild-type SLT-1 A₁ chain (15 µM) as a function of increasing salt concentrations.

Figure 4. The interaction of the A₁ chain of SLT-1 with the ribosomal stalk protein P2 and the C-terminal peptide SDDDMGFGLFD also involves hydrophobic residues within the A₁ chain.

(A) Bait vectors expressing either a catalytically inactive variant of the wild-type SLT-1 A₁ domain (CIA₁) or one of the hydrophobic mutants were co-transformed in the yeast strain AH109 with a prey vector expressing ribosomal protein P2. The transformed yeast cells were plated on SD agar −Trp/−Leu. The resulting yeast colonies were grown overnight, and spotted (10 µl) as 10-fold serial dilutions onto SD medium lacking Trp and Leu to select for the presence of each plasmid followed by spotting on SD media lacking Trp, Leu, and His to select for interacting partners. (B) SPR profiles (plotted at 15 µM) demonstrate that hydrophobic mutants F226A and S235A in the SLT-1 A₁ chain have a minor effect on the binding to the conserved peptide SDDDMGFGLFD and the SLT-1 V191A and L233A A₁ chain mutants cause a drastic decrease in binding. Experiments were performed in triplicate.

Figure 5. Arginine-to-alanine and hydrophobic variants of SLT-1 A₁ that bind weakly to the monomeric conserved C-terminal motif display altered ribosome-inactivating activities when compared to the wild-type A₁ chain.

Eight ten-fold serial dilutions of the wild-type and each charge and hydrophobic A₁ chain variant was dispensed into an in vitro transcription and translation-coupled rabbit reticulocyte lysate system to monitor their ability to block protein synthesis (methods section). The level of in vitro protein synthesis was assessed by measuring the incorporation of [³⁵S]-methionine into the reporter protein luciferase during its synthesis. The expression of radiolabeled luciferase (arrow) was then resolved by SDS-PAGE and quantified using a phosphorimager. The addition of PBS alone (- lane) was used as a control.

Figure 6. Primary and tertiary structural comparisons between SLT-1 and SLT-2 highlighting the conservation of important ribosomal stalk peptide contact sites.

(A) Left Panel - Surface rendering of the SLT-1 A₁ chain (PDB# 1DM0) depicting the cationic (blue) and hydrophobic (yellow) residues essential for optimal binding to the conserved stalk peptide SDDDMGFGLFD as well as Arg-188 (light blue) which has a modest effect on peptide binding. Right Panel – Structure as shown in the left panel rotated by 140°, highlighting the catalytic residues in green. (B) Three-dimensional stick structures of SLT-1 (left panel), SLT-2 (PDB# 1R4P; middle panel), and the structural alignment of the two toxins (right panel). Cationic residues are labeled in blue and red, while hydrophobic residues are labeled in yellow and orange for SLT-1 and SLT-2 respectively. (C) Primary amino acid sequence alignment of SLT-1 and SLT-2 within residues 158 and 250. Catalytic residues are highlighted in green and cationic and hydrophobic residues in blue and yellow, respectively. Surface and stick renderings and alignments were performed using the The PyMOL Molecular Graphics System (Version 1.3, Schrödinger, LLC), whereas amino acid sequences were aligned using BioEdit software .