Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin.

Yijun Zhou,Xiao-Ping Li,John E Mclaughlin,Nilgun E Tumer,Jennifer N Kahn

doi:10.1042/bsr20192022

Abstract

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.

Highlights

Ricin, produced by the castor bean plant, is a type II ribosome inactivating protein (RIP), which consists of a ricin toxin A chain (RTA) and a ricin toxin B chain (RTB) connected by a disulfide bond
To determine the relative contribution of the hydrophobic residues to the depurination activity and toxicity of RTA, Tyr183, Leu207, Leu232, Phe240, Val242, Ile247, P250 and Ile251, which interact with the last six residues of P proteins [33,34] (Figure 1A,B) were each replaced with alanine on the mature RTA (mRTA) and cloned into the low copy yeast expression vector pRS415 under the control of the GAL1 promoter
These data showed that the activity of Y183A, L207A, L232A and F240A was reduced compared with WT RTA and L232A was the least toxic and the least active hydrophobic mutant

Summary

Introduction

Ricin, produced by the castor bean plant, is a type II ribosome inactivating protein (RIP), which consists of a ricin toxin A chain (RTA) and a ricin toxin B chain (RTB) connected by a disulfide bond. RTB is a lectin that can bind to glycoprotein or glycolipids on the surface of the cell membrane and facilitate endocytosis of the toxin [1]. The disulfide bond is reduced in the ER, releasing RTA from RTB [2]. Other type II RIPs such as abrin and Shiga toxin have an enzymatically active A chain linked to a B chain [4]. Type I RIPs, such as pokeweed antiviral protein (PAP), saporin, gelonin and trichosanthin (TCS), contain only one enzymatically active chain [4]. RIPs depurinate a conserved adenine (A4324 in rat) in the α-sarcin/ricin loop (SRL) of the large rRNA [5], inhibiting interaction of the SRL with the elongation factors [6,7,8]

Methods

Results

Conclusion