Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding

Abstract

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 RTA⁻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 RTA⁻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.

Keywords: P protein interaction; SRL depurination; peptide inhibition; ribosomal P stalk; ricin A chain.

Figure 1

The sequence of the peptides corresponding to the C-terminal end of P proteins.

Figure 2

The interaction curves (a) and the steady state affinity fitting (b) of the peptide—Ricin A chain (RTA) interaction. The K_D was determined using Biacore T200. The untagged recombinant RTA was immobilized on a CM5 chip by amine coupling at about 2422 RU. The reference surface was activated and blocked. The peptides were passed over the surface at 6.2 µM (red), 18.5 µM (green), 55.6 µM (dark blue), 166.7 µM (magenta), and 500 µM (light blue).

Figure 3

The interaction curves of P11 (a–c) and P4 (d–f) with wild-type (WT) RTA and the R193A, R235A, R189A, and R234A point mutants. The interactions were analyzed by Biacore T200 using the same conditions as in Figure 2, except N-terminally His-tagged wild-type RTA (10×His-RTA) or 10×His-tagged RTA mutants were captured on an NTA chip at around 2100 RU. Flow cell 1 (Fc1) was used as control, R235A or R234A was captured on Fc2, R193A or R189A was captured on Fc3, and wild type (WT)-RTA was captured on Fc4. The P11 or P4 were passed over the surface at a concentration of 166.7 µM. The signals were normalized to the chip density of WT-RTA.

Figure 4

The interaction of peptides mimicking the last five amino acids (P5) of the P proteins (a,c) and the penultimate five amino acids (P5b) with RTA (b,d). The K_D was determined by Biacore T200 using the same conditions as in Figure 2. The binding sensorgrams are shown in (a,b) and the fitting is shown in (c,d).

Figure 5

P6 competes with the ribosome for binding to RTA. The A-B-A capability of Biacore 8K was used for the competition analysis. RTA was immobilized on a CM5 chip at 4000 RU by amine coupling. The P6 was passed over the RTA at indicated concentrations for 1 min and then yeast ribosomes (20 nM) were injected over the surface together with the same concentrations of P6 for another 2 min at a flow rate of 30 µL per min. The ribosome binding levels were determined at 5 s before the end of the injection. The surface was regenerated by three one-minute injections of 2 M NaCl and one injection of running buffer with 2% DMSO. The data are expressed as average ± SD from four replicates.

Figure 6

Inhibition of depurination activity of RTA by P11. The depurination levels were determined by qRT-PCR. (a) Yeast ribosomes were used at 60 nM and RTA was used at 1.0 nM. (b) Rat liver ribosomes were used at 60 nM and RTA was used at 0.2 nM. Different concentrations of P11 and RTA were mixed first and the reaction was started by adding ribosomes. The reaction was incubated at the room temperature for 5 min and was stopped by adding 2×RNA extraction buffer. The RNA was purified and the depurination levels were determined by qRT-PCR. The depurination level of the reaction without toxin was set as 100%. The depurination levels were calculated and plotted as percent of no toxin control. Experiments were conducted four to six times and the data were fit with the Michaelis–Menten equation using Origin Pro 9.1. IC₅₀: the half maximal inhibitory concentration. I_max: maximal inhibition.