A Two-Step Binding Model Proposed for the Electrostatic Interactions of Ricin A Chain with Ribosomes

Abstract

Ricin is a ribosome inactivating protein that catalytically removes a universally conserved adenine from the α-sarcin/ricin loop (SRL) of the 28S rRNA. We recently showed that ricin A chain (RTA) interacts with the P1 and P2 proteins of the ribosomal stalk to depurinate the SRL in yeast. Here we examined the interaction of RTA with wild type and mutant yeast ribosomes deleted in the stalk proteins by surface plasmon resonance. The interaction between RTA and wild type ribosomes did not follow a single step binding model, but was best characterized by two distinct types of interactions. The AB1 interaction had very fast association and dissociation rates, was saturable and required an intact stalk, while the AB2 interaction had slower association and dissociation rates, was not saturable and did not require the stalk. RTA interacted with the mutant ribosomes by a single type of interaction, which was similar to the AB2 interaction with the wild type ribosomes. Both interactions were dominated by electrostatic interactions and the AB1 interaction was stronger than the AB2 interaction. Based on these results we propose a two-step interaction model. The slow and ribosomal stalk nonspecific AB2 interactions concentrate the RTA molecules on the surface of the ribosome. The AB2 interactions facilitate the diffusion of RTA towards the stalk and promote the faster, more specific AB1 interactions with the ribosomal stalk. The electrostatic AB1 and AB2 interactions work together allowing RTA to depurinate the SRL at a much higher rate on the intact ribosomes than on the naked 28S rRNA.Ricin is a ribosome inactivating protein (RIP) isolated from the castor bean plant, Ricinus communis that consists of a Ricin Toxin A chain (RTA) and a galactose-binding B chain (RTB). RTA and RTB are linked by a disulfide bond between the Cys 259 near the C-terminus of RTA and the Cys 4 of RTB (1). The holotoxin is not active on ribosomes (2-6). The enzymatic activity of ricin is due to RTA, which is an N-glycosidase that specifically cleaves the Nglycosidic bond at A4324 of the 28S rRNA of rat ribosomes (7) and inhibits the elongation factor dependent ribosomal functions (8-10). RTA can also cleave the A2660 in naked 23S rRNA from E. coli but not the ribosomes from E. coli (11)(12)(13)(14). Both A4324 of rat 28S rRNA and A2660 of E. coli 23S rRNA are located at a universally conserved rRNA stem loop called the α-sarcin/ricin loop (SRL) named after the toxins (9). The SRL is the site where elongation factors interact with the ribosome and is involved in peptide translocation during protein synthesis (15). RTA depurinates the 28S rRNA on rat ribosomes about 5000 times faster than the naked rat 28S rRNA (7), indicating that the conformation of the rRNA on intact ribosomes and the ribosomal proteins play an important role in ribosome depurination by ricin.The α-sarcin/ricin loop (SRL) is located on the 60S subunit in close proximity to the ribosomal stalk, which is a lateral flexible structure on the large subunit. The stalk region was ...

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

“…Here, we present the binding kinetics and affinity of the interactions between RTA and the native stalk isolated from the yeast, S. cerevisiae. Our results show that RTA interacts with the isolated stalk pentamer in a simple 1:1 interaction model, consistent with our previously proposed model (40). We further show that the association rate of RTA with the stalk pentamer is higher than with either trimer, demonstrating that multiple copies of stalk proteins accelerate the recruitment of RTA to the ribosome for depurination.…”

“…The C-terminal His-tagged RTA was purified from Escherichia coli as described previously (40). Both proteins showed a single band on SDS-PAGE by Coomassie Blue staining and by immunoblot analysis and were active in in vitro translation inhibition and ribosome depurination assays (40).…”

“…characterize the RTA and stalk complex interaction, we examined the binding kinetics of the interaction between RTA and the TH199 complex using a Biacore 3000 under the same conditions that were previously used to study the interaction of RTA with ribosomes (40). As shown in Fig.…”

“…Our previous work, using intact ribosomes, showed that RTA interacts with the ribosomal stalk to access its substrate, the SRL in yeast, and that this interaction is important for the cytotoxicity of RTA (39). The interaction of RTA with ribosomes did not follow a simple 1:1 interaction model and was characterized by a two-step binding model (40). According to this model, the slower, nonspecific interactions concentrate RTA molecules on the surface of ribosomes and facilitate faster, more specific interactions with the stalk (40).…”

“…The interaction of RTA with ribosomes did not follow a simple 1:1 interaction model and was characterized by a two-step binding model (40). According to this model, the slower, nonspecific interactions concentrate RTA molecules on the surface of ribosomes and facilitate faster, more specific interactions with the stalk (40). Based on this model, we predicted that there should be only one type of interaction between RTA and the isolated stalk complex.…”