Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome

Abstract

The A1 subunits of Shiga toxin 1 (Stx1A1) and Shiga toxin 2 (Stx2A1) interact with the conserved C termini of ribosomal-stalk P-proteins to remove a specific adenine from the sarcin/ricin loop. We previously showed that Stx2A1 has higher affinity for the ribosome and higher catalytic activity than Stx1A1. To determine if conserved arginines at the distal face of the active site contribute to the higher affinity of Stx2A1 for the ribosome, we mutated Arg172, Arg176, and Arg179 in both toxins. We show that Arg172 and Arg176 are more important than Arg179 for the depurination activity and toxicity of Stx1A1 and Stx2A1. Mutation of a single arginine reduced the depurination activity of Stx1A1 more than that of Stx2A1. In contrast, mutation of at least two arginines was necessary to reduce depurination by Stx2A1 to a level similar to that of Stx1A1. R176A and R172A/R176A mutations eliminated interaction of Stx1A1 and Stx2A1 with ribosomes and with the stalk, while mutation of Arg170 at the active site reduced the binding affinity of Stx1A1 and Stx2A1 for the ribosome, but not for the stalk. These results demonstrate that conserved arginines at the distal face of the active site are critical for interactions of Stx1A1 and Stx2A1 with the stalk, while a conserved arginine at the active site is critical for non-stalk-specific interactions with the ribosome. Arginine mutations at either site reduced ribosome interactions of Stx1A1 and Stx2A1 similarly, indicating that conserved arginines are critical for ribosome interactions but do not contribute to the higher affinity of Stx2A1 for the ribosome. Shiga toxin (Stx)-producing Escherichia coli (STEC) is an emerging foodborne and waterborne pathogen responsible for hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC), which are the leading causes of acute renal failure and mortality in children in the United States (1). STEC serotypes, such as E. coli O157:H7, are associated with severe disease (2). Antibiotics are known to exacerbate the disease symptoms, and at present, there are no FDA-approved vaccines or therapeutics against STEC infection (3-5). STEC produces a family of structurally and functionally related virulence factors called Shiga toxins, the most predominant ones being Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) (6). Stx2 and Stx1 have one prototype (Stx1a and Stx2a) and several subtypes. Stxs are type II ribosome-inactivating proteins (RIPs) with a catalytically active A subunit attached to a pentamer of B subunits. The B subunits facilitate the endocytosis of the toxins into the cell by binding to a common receptor globotriaosylceramide (GB3 or CD77). The toxin travels in a retrograde manner from the endosome to the endoplasmic reticulum (ER) via the Golgi network (7). In order to intoxicate the cell, the A subunit is cleaved into the A1 fragment and the A2 fragment, which remain together via a disulfide bond. After reduction of the disulfide bond, the A1 fragment is translocated into the cytosol from the ER, where it refolds into an active confor...

Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins

“…Previous results indicated that the A1 subunit of Stx2 interacts with the ribosome with higher affinity than the A1 subunit of Stx1 [14]. Conserved 1 subunits for the rib arginines at the P-protein stalk binding site did not contribute to the differences in the affinity of the A osome [32]. We show here that Stx2 holotoxin also interacts with the ribosome with higher affinity than Stx1 holotoxin (Figure 4).…”

“…Our recent study showed that point mutations at Arg172 or Arg176 located on the opposite face of the active site (Figure 1 and Figure 3) disrupted the ribosome and ribosomal stalk interaction of Stx1A1 and Stx2A1, but did not affect their depurination activity on RNA, indicating that their active sites were intact and were located on the opposite face of the ribosome binding site [32]. Figure 3 shows the structure of Stx holotoxins in comparison with ricin.…”

Functional Assays for Measuring the Catalytic Activity of Ribosome Inactivating Proteins

“…The base change at the depurination site was detected using a reverse primer with adenine at the 3′ terminus and the specificity for detection was enhanced by the inclusion of a secondary mismatch two base pairs 5′ to the adenine. The modified qRT-PCR assay provided a sensitive and highly reproducible measure of the extent of depurination by ricin and Stxs in mammalian cells [ 31 , 32 , 37 , 40 ] and in yeast [ 31 , 34 , 36 , 37 ] in vivo.…”

“…The contribution of toxin-ribosome interactions to the depurination activity of RIPs was determined by comparing the activity of mutants on ribosomes and an SRL mimic RNA, since depurination activity on rRNA does not require ribosome binding [ 31 , 34 , 35 , 36 , 37 ]. Using the qRT-PCR and the enzymatically-coupled adenine detection assay, mutants defective in ribosome binding, but not catalytic activity were identified and ribosome-binding site of RTA and Stxs were mapped to the distal face of the active site [ 31 , 34 , 35 , 36 , 37 ]. These assays showed that the interaction of Stxs and RTA with the stalk proteins has a profound effect on their catalytic efficiency towards the SRL [ 34 , 36 , 37 , 89 ].…”

“…Assays that directly measure depurination activity for toxin detection and mitigation procedures were recently reviewed [ 29 , 30 ]. Here, we specifically review the assays, which identified differences in the activity of Stxs [ 31 ], differences in ribosome interactions of RTA [ 22 , 32 , 33 , 34 , 35 , 36 ], Stxs [ 21 , 22 , 37 ], TCS [ 23 , 26 ], and PAP [ 24 , 25 ], provided information about the downstream consequences of depurination, such as the ribotoxic stress response and apoptosis [ 38 , 39 , 40 ], allowed the use of RIPs as tools to probe the structure and function of the ribosomal stalk [ 41 ], provided information about the functional divergence of the ribosomal stalk P1-P2 dimers [ 42 ] and were useful in the search for RIP inhibitors [ 43 , 44 , 45 , 46 , 47 , 48 ]. We discuss the advantages and limitations of each method and explain how more sensitive assays can distinguish the differences in depurination activity and provide information about the kinetics and the mechanism of the depurination reaction.…”