Pentameric Organization of the Ribosomal Stalk Accelerates Recruitment of Ricin A Chain to the Ribosome for Depurination

Li, Xiaoping; Grela, Przemysław; Krokowski, Dawid; Tchórzewski, Marek; Tumer, Nilgun E.

doi:10.1074/jbc.m110.171793

Cited by 32 publications

(61 citation statements)

References 48 publications

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“…To compare the interaction of Stx1A1 and Stx2A1 with the ribosome, the untagged A1 subunits were immobilized on a CM5 chip of the Biacore T200 instrument at 1,692 RU and 1,672 RU, respectively, using amine coupling, and their interaction with yeast ribosomes was measured by single-cycle kinetic analysis by passing ribosomes over the surface at different concentrations. Since the surface is not regenerated between injections in the single-cycle analysis, multiple samples can be analyzed in a shorter period of time, allowing comparison of levels of binding under identical conditions (38). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, the interaction of Stx1 and Stx2 with either the ribosome or the ribosomal stalk complex has not been investigated. To investigate the basis for the higher activity of Stx2A1, we compared the interactions of the A1 subunits with yeast ribosomes and with the isolated ribosomal P-protein stalk pentamer from yeast (38). Our previously reported results indicated that the interaction of RTA with ribosomes did not fit a simple 1:1 interaction model (32).…”

Section: Discussionmentioning

confidence: 99%

“…The A1 chain of Stx1 interacts with the conserved C termini of P proteins (33,34). To determine if the higher level of binding of Stx2A1 to the ribosome is due to a higher level of binding to the ribosomal P-protein stalk complex, we examined the interaction of the A1 subunits with the pentameric stalk complex from yeast (38). We captured the 10ϫHis-tagged Stx1A1 and Stx2A1 proteins on an NTA chip at the same level and passed the purified stalk pentamer complex over the surface.…”

Section: Fig 2 (A) Coomassie Blue Staining Of Purified 10ϫhis-tagged mentioning

confidence: 99%

“…All stalk P proteins contain a highly conserved motif at their C termini, which is involved in the recruitment of external factors to the ribosome (37). We showed that the P proteins of the ribosomal stalk are essential for the cytotoxicity of ricin toxin A chain (RTA) and that the ribosomal stalk is the docking site where RTA interacts with the ribosome (31,32,38). The catalytic A1 subunit of Stx1 was shown to interact with the conserved C-terminal motif of the P proteins (33).…”

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confidence: 99%

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The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

Basu

Kahn

et al. 2016

Infect Immun

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Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1. Shiga toxin-producing Escherichia coli (STEC) is an emerging foodborne and waterborne pathogen. STEC infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), with potentially lethal consequences (1). Due to a very low infectious dose and ease of person-to-person spread, STEC infection is the leading cause of death from foodborne bacterial infection in children (2). Presently, there are no postexposure therapeutics or vaccines available for STEC infection. Due to recent outbreaks of E. coli O157:H7 in the United States and the emergence of highly virulent new strains, such as E. coli O104:H4, which caused the deadliest HUS outbreak in Germany in 2011, STEC remains a major challenge for food safety and public health (3-6).The primary virulence factors of STEC, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), are AB 5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits and are known as type II ribosome-inactivating proteins (RIPs). The A subunits of Stx1 and Stx2 consist of the catalytically active A1 (residues 1 to 251 in Stx1 and 1 to 250 in Stx2) and A2 (residues 252 to 293 in Stx1 and 251 to 297 in Stx2) chains, which are cleaved by the protease furin and kept together by a disulfide bond (7). The B subunits bind ...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Fig 2 (A) Coomassie Blue Staining Of Purified 10ϫhis-tagged mentioning

confidence: 99%

mentioning

confidence: 99%

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The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

Basu

Kahn

et al. 2016

Infect Immun

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“…The most prominent feature of the eukaryotic stalk proteins is the highly conserved motif present in the C-terminal part of the uL10 and P1/P2 proteins, consisting of a stretch of highly acidic and hydrophobic amino acids involved in interaction with translational GTPases (trGTPases) and RIPs (21). We established that the ribosomal stalk is the primary docking site for the ricin A chain (RTA) on the ribosome (13) and showed that multiplication of P-proteins is a critical factor accelerating the recruitment of RTA to the ribosome (22). Using the yeast model, we showed that the two P1A/P2B and P1B/P2A stalk dimers on the ribosome do not contribute equally to the interaction of ribosomes with RTA (23).…”

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confidence: 99%

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

Basu

Kahn

et al. 2016

Infect Immun

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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...

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Phosphorylation of the conserved C‐terminal domain of ribosomal P‐proteins impairs the mode of interaction with plant toxins

et al. 2021

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The ribosome is subjected to post‐translational modifications, including phosphorylation, that affect its biological activity. Among ribosomal elements, the P‐proteins undergo phosphorylation within the C terminus, the element which interacts with trGTPases or ribosome‐inactivating proteins (RIPs); however, the role of phosphorylation has never been elucidated. Here, we probed the function of phosphorylation on the interaction of P‐proteins with RIPs using the ribosomal P1‐P2 dimer. We determined the kinetic parameters of the interaction with the toxins using biolayer interferometry and microscale thermophoresis. The results present the first mechanistic insight into the function of P‐protein phosphorylation, showing that introduction of a negative charge into the C terminus of P1‐P2 proteins promotes α‐helix formation and decreases the affinity of the P‐proteins for the RIPs.

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Pentameric Organization of the Ribosomal Stalk Accelerates Recruitment of Ricin A Chain to the Ribosome for Depurination

Cited by 32 publications

References 48 publications

The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

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

Phosphorylation of the conserved C‐terminal domain of ribosomal P‐proteins impairs the mode of interaction with plant toxins

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