Abstract
Ricin toxin A chain (RTA) binds to stalk P-proteins to reach the α–sarcin/ricin loop (SRL) where it cleaves a conserved adenine. Arginine residues at the RTA/RTB interface are involved in this interaction. To investigate the individual contribution of each arginine, we generated single, double and triple arginine mutations in RTA. The R235A mutation reduced toxicity and depurination activity more than any other single arginine mutation in yeast. Further reduction in toxicity, depurination activity and ribosome binding was observed when R235A was combined with a mutation in a nearby arginine. RTA interacts with the ribosome via a two-step process, which involves slow and fast interactions. Single arginine mutations eliminated the fast interactions with the ribosome, indicating that they increase the binding rate of RTA. Arginine residues form a positively charged patch to bind to negatively charged residues at the C-termini of P-proteins. When electrostatic interactions conferred by the arginines are lost, hydrophobic interactions are also abolished, suggesting that the hydrophobic interactions alone are insufficient to allow binding. We propose that Arg235 serves as an anchor residue and cooperates with nearby arginines and the hydrophobic interactions to provide the binding specificity and strength in ribosome targeting of RTA.
Highlights
The structural analysis showed that ricin toxin A chain (RTA) interacts with P-proteins via hydrophobic rather than electrostatic interactions[27], we show here that arginines at the RTA/ricin toxin B chain (RTB) interface contribute to the fast electrostatic interactions with the ribosome
To identify the contribution of each arginine at the RTA/RTB interface to the toxicity of the toxin, Arg[189], Arg[191], Arg[193], Arg[196], Arg[197], Arg[234] and Arg[235] were mutated to alanine and their cytotoxicity was analyzed in yeast in comparison to wild type (WT) RTA
In the crystal structure P6 peptide [GFGLFD] docks into a pocket formed by six hydrophobic residues (Tyr[183], Leu[207], Phe[240], Ile[247], Pro[250] and Ile251) and five polar residues (Gln[182], Ser[203], Gln[233], Arg[234] and Arg235) in RTA27
Summary
We previously showed that R189A/ R234A and R193A/R235A double mutations affected the electrostatic interactions of RTA with the ribosome[14] and reduced the depurination activity and toxicity of RTA To address the relative importance of each arginine at the RTA/RTB interface as an anchor for the CTD of ribosomal stalk proteins, we generated RTA constructs with single, double and triple mutations. These mutants were assessed for their ability to bind ribosomes, to depurinate the SRL and to cause toxicity in yeast. We present the first evidence that Arg[235] serves as a key interacting residue and cooperates with nearby arginines to allow RTA to bind to the stalk with fast kinetics to achieve binding specificity and speed in depurinating the SRL
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