RESUMEN
Ricin toxin A subunit (RTA) is the catalytic subunit of ricin, which depurinates an adenine from the sarcin/ricin loop in eukaryotic ribosomes. There are no approved inhibitors against ricin. We used a new strategy to disrupt RTA-ribosome interactions by fragment screening using surface plasmon resonance. Here, using a structure-guided approach, we improved the affinity and inhibitory activity of small-molecular-weight lead compounds and obtained improved compounds with over an order of magnitude higher efficiency. Four advanced compounds were characterized by X-ray crystallography. They bind at the RTA-ribosome binding site as the original compound but in a distinctive manner. These inhibitors bind remotely from the catalytic site and cause local conformational changes with no alteration of the catalytic site geometry. Yet they inhibit depurination by ricin holotoxin and inhibit the cytotoxicity of ricin in mammalian cells. They are the first agents that protect against ricin holotoxin by acting directly on RTA.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Ribosomas/efectos de los fármacos , Ricina/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Chlorocebus aethiops , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Ricina/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Células VeroRESUMEN
Ricin toxin A subunit (RTA) removes an adenine from the universally conserved sarcin/ricin loop (SRL) on eukaryotic ribosomes, thereby inhibiting protein synthesis. No high affinity and selective small molecule therapeutic antidotes have been reported against ricin toxicity. RTA binds to the ribosomal P stalk to access the SRL. The interaction anchors RTA to the P protein C-termini at a well-defined hydrophobic pocket, which is on the opposite face relative to the active site. The RTA ribosome binding site has not been previously targeted by small molecule inhibitors. We used fragment screening with surface plasmon resonance to identify small molecular weight lead compounds that bind RTA and defined their interactions by crystallography. We identified five fragments, which bound RTA with mid-micromolar affinity. Three chemically distinct binding fragments were cocrystallized with RTA, and crystal structures were solved. Two fragments bound at the P stalk binding site, and the third bound to helix D, a motif distinct from the P stalk binding site. All fragments bound RTA remote from the catalytic site and caused little change in catalytic site geometry. Two fragments uniquely bound at the hydrophobic pocket with affinity sufficient to inhibit the catalytic activity on eukaryotic ribosomes in the low micromolar range. The binding mode of these inhibitors mimicked the interaction of the P stalk peptide, establishing that small molecule inhibitors can inhibit RTA binding to the ribosome with the potential for therapeutic intervention.
Asunto(s)
Ricina , Sitios de Unión , Péptidos/metabolismo , Unión Proteica , Ribosomas/metabolismo , Ricina/metabolismoRESUMEN
Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of HAC1u (u for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants. An active-site mutant with very low depurination activity, which bound ribosomes as WT RTA, did not inhibit HAC1u mRNA splicing. A ribosome-binding mutant, which showed reduced binding to ribosomes but retained depurination activity, inhibited HAC1u mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. The ribosome-binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of HAC1u mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that ribosomes play a role in splicing of HAC1u mRNA. We show that HAC1u mRNA is associated with ribosomes and does not get processed on depurinated ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits HAC1u mRNA splicing through its depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes HAC1u mRNA that is associated with ribosomes.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Purinas/metabolismo , Empalme del ARN/genética , Proteínas Represoras/metabolismo , Ribosomas/metabolismo , Ricina/toxicidad , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estrés del Retículo Endoplásmico/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Mutación/genética , Multimerización de Proteína/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Procesamiento Postranscripcional del ARN/genética , Empalme del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genéticaRESUMEN
Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruple mutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.
Asunto(s)
Ribosomas/química , Ricina/química , Saccharomyces cerevisiae/química , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , Leucina , Ribosomas/genética , Ribosomas/metabolismo , Ricina/genética , Ricina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Ricin A chain (RTA) depurinates the sarcin/ricin loop (SRL) by interacting with the C-termini of the ribosomal P stalk. The ribosome interaction site and the active site are located on opposite faces of RTA. The interaction with P proteins allows RTA to depurinate the SRL on the ribosome at physiological pH with an extremely high activity by orienting the active site towards the SRL. Therefore, if an inhibitor disrupts RTAâ»ribosome interaction by binding to the ribosome binding site of RTA, it should inhibit the depurination activity. To test this model, we synthesized peptides mimicking the last 3 to 11 amino acids of P proteins and examined their interaction with wild-type RTA and ribosome binding mutants by Biacore. We measured the inhibitory activity of these peptides on RTA-mediated depurination of yeast and rat liver ribosomes. We found that the peptides interacted with the ribosome binding site of RTA and inhibited depurination activity by disrupting RTAâ»ribosome interactions. The shortest peptide that could interact with RTA and inhibit its activity was four amino acids in length. RTA activity was inhibited by disrupting its interaction with the P stalk without targeting the active site, establishing the ribosome binding site as a new target for inhibitor discovery.
Asunto(s)
Péptidos/metabolismo , Ribosomas/metabolismo , Ricina/metabolismo , Unión ProteicaRESUMEN
Ribosome-inactivating proteins (RIPs) are potent toxins that inactivate ribosomes by catalytically removing a specific adenine from the α-sarcin/ricin loop (SRL) of the large rRNA. Direct assays for measuring depurination activity and indirect assays for measuring the resulting translation inhibition have been employed to determine the enzyme activity of RIPs. Rapid and sensitive methods to measure the depurination activity of RIPs are critical for assessing their reaction mechanism, enzymatic properties, interaction with ribosomal proteins, ribotoxic stress signaling, in the search for inhibitors and in the detection and diagnosis of enteric infections. Here, we review the major assays developed for measuring the catalytic activity of RIPs, discuss their advantages and disadvantages and explain how they are used in understanding the catalytic mechanism, ribosome specificity, and dynamic enzymatic features of RIPs.
Asunto(s)
Bioensayo , Proteínas Inactivadoras de Ribosomas/metabolismo , Animales , CatálisisRESUMEN
Clostridium difficile is a significant concern as a nosocomial pathogen, and genetic tools are important when analyzing the physiology of such organisms so that the underlying physiology/pathogenesis of the organisms can be studied. Here, we used TargeTron to investigate the role of selenoproteins in C. difficile Stickland metabolism and found that a TargeTron insertion into selD, encoding the selenophosphate synthetase that is essential for the specific incorporation of selenium into selenoproteins, results in a significant growth defect and a global loss of selenium incorporation. However, because of potential polar effects of the TargeTron insertion, we developed a CRISPR-Cas9 mutagenesis system for C. difficile. This system rapidly and efficiently introduces site-specific mutations into the C. difficile genome (20-50% mutation frequency). The selD CRISPR deletion mutant had a growth defect in protein-rich medium and mimicked the phenotype of a generated TargeTron selD mutation. Our findings suggest that Stickland metabolism could be a target for future antibiotic therapies and that the CRISPR-Cas9 system can introduce rapid and efficient modifications into the C. difficile genome.
Asunto(s)
Clostridioides difficile/metabolismo , Edición Génica/métodos , Selenoproteínas/metabolismo , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Clostridioides difficile/genética , Electroforesis en Gel de Poliacrilamida , Genes Bacterianos/genética , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Selenio/metabolismo , Selenoproteínas/genéticaRESUMEN
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.
Asunto(s)
Secuencia Conservada , Proteínas de Escherichia coli/metabolismo , Complejos Multienzimáticos/metabolismo , Prefenato Deshidratasa/metabolismo , Ribosomas/metabolismo , Saccharomyces/metabolismo , Toxinas Shiga/metabolismo , Animales , Sitios de Unión , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Modelos Moleculares , Complejos Multienzimáticos/genética , Mutación , Plásmidos , Prefenato Deshidratasa/genética , Unión Proteica , Conformación Proteica , Subunidades de Proteína , ARN de Hongos/metabolismo , Ratas , Ribosomas/química , Saccharomyces/genética , Toxinas Shiga/químicaRESUMEN
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.