**1. Introduction**

Ricin (E.C. 3.2.2.22), produced by the castor bean (*Ricinus communis*), belongs to a group of toxic proteins called ribosome-inactivating proteins (RIPs) that include major human pathogens, such as *Escherichia coli* and *Shigella* producing Shiga toxins (Stxs). They are classified as category B agents of national security and public health risk with potential for significant morbidity and mortality. Currently, no U.S. Food and Drug Administration-approved vaccines or therapeutics exist to protect against ricin, Stxs, or any other RIP. The RIPs cleave a universally conserved adenine from the sarcin/ricin loop (SRL) on the large rRNA, inhibiting protein synthesis and inducing cell death [1–4]. Ricin A chain (RTA) interacts with the P proteins of the ribosomal stalk to depurinate the SRL [5]. Several other RIPs, including Shiga toxins [6–8], also interact with the conserved C-terminal domain (CTD) of P proteins in order to access the SRL [9–11].

In eukaryotes, the P stalk is a pentameric protein complex composed of two P1/P2 dimers that bind to the C-terminus of the uL10 (previously P0) protein, while the N-terminal domain of uL10

anchors the stalk on the large subunit of the ribosome [12–14]. The P stalk and the SRL are part of the GTPase-associated center (GAC) in the large subunit [13,15]. The unique feature of all P proteins is the 11 C-terminal amino acids, which are identical in all eukaryotes and have a disordered structure [16]. The CTD of P proteins selectively recognizes translational GTPases, such as the initiation factor 5B (eIF5B) and the elongation factors eEF-2/EFG and eEF1 α/EFTu, and recruits them to the SRL [17–20].

The interaction of RTA with P proteins is critical for ribosome binding, depurination of the SRL, and toxicity of RTA in the yeas<sup>t</sup> *Saccharomyces cerevisiae* and in human cells [5,21]. We showed that the ribosome binding site and the active site are located on opposite faces of RTA and based on these results we proposed a molecular model for depurination of the SRL by RTA [22]. According to this model, RTA is concentrated around the ribosome by electrostatic interactions [23]. The P stalk interacts with RTA and stalk binding stimulates the catalysis of depurination by orienting the active site of RTA towards the SRL [22]. The interaction with the P proteins allows RTA to depurinate the SRL on the ribosome at physiological pH with an extremely high catalytic activity, while at this pH RTA is not active on the naked RNA [22,24]. Based on this model, we predict that if an inhibitor disrupts the interaction of RTA with the ribosome by binding to the ribosome binding site of RTA, it should be able to inhibit the depurination activity of RTA.

Until now, no inhibitor targeting the ribosome binding site of RTA has been reported. A 17-mer peptide mimicking the CTD of the human ribosomal stalk P2 protein was shown to inhibit the activity of the A1 subunit of Shiga toxin 1 (Stx1A1) in an in vitro translation assay [6]. Calmodulin-tagged peptides corresponding to the last 11 and 17 residues of the human P2 protein could pull down Stx1A1 and RTA, but not a peptide corresponding to the last 7 residues of human P2 [6]. The 11-mer peptide (P11), S105DDDMGFGLFD115, contains a negatively charged acidic motif "D106 D107 D108" at its N-terminus and a hydrophobic "F111GLFD115" motif at its C-terminus. The pull down and binding studies showed that both motifs are important for the interaction of Stx1A1 with P11 [7].

In *S. cerevisiae*, P1/P2 proteins have diverged into P1 α/P2β and P1β/P2 α [25]. The P1 α/P2β dimer could inhibit the depurination activity of Stx1A1 and Stx2A1 on ribosomes isolated from a yeas<sup>t</sup> mutant in which the binding site of the P protein dimers on the P0 protein had been deleted, suggesting that P1 α/P2β can bind to the toxins and prevent them from depurinating ribosomes [8]. Although the amino acid sequences of the A1 subunit of Stx1 and Stx2 are only 21% and 20% identical to RTA, respectively, they are structurally and functionally very similar to RTA [26]. Recently, the structure of RTA together with the last six amino acids of P proteins was resolved by two different groups using different strategies (PDB IDs: 5GU4 and 5DDZ) [27,28]. Although 9-mer, 11-mer [27], or 10-mer [28] P stalk peptides were used in the studies, the structures resolved by both groups showed that only the last six residues interact with RTA. The last six residues (G110FGLFD115) bind to a hydrophobic pocket on RTA in a unique conformation. Neither group could visualize the N-terminal end of the peptide that contains the negatively charged (D106 D107 D108) motif. However, the two groups drew different conclusions. One group postulated that this motif contributes to the interaction because GST-tagged peptides, which contained the D106 D107 D108 motif, showed higher affinities for RTA than those without this motif [27]. In contrast, the other group concluded that the D106 D107 D108 motif does not contribute to the RTA interaction because they did not observe any difference in the pull-down experiments with His-tagged RTA when this motif was mutated or deleted [28]. We previously showed that arginines at the RTA/RTB interface contribute to fast electrostatic interactions with the CTD of the P proteins, indicating that the negatively charged motif plays an important role in the interaction of RTA with the ribosome [29].

Although the active site of RTA has been explored extensively as a target for antidotes, the interaction of RTA with ribosomes has not been previously examined as a potential drug target. To better understand the recognition mechanism of the P protein CTD by RTA and to define the minimal length of a peptide that can bind RTA and inhibit its activity, we measured the interaction of peptides corresponding to the last 3 to 11 amino acids of human P proteins with RTA and examined their ability to inhibit the depurination activity of RTA. We discuss the relationship between the affinity of the peptides and their inhibitory activity. Our results establish the ribosome binding site of RTA as a new target for inhibitor discovery. Since Stxs also bind to the P protein CTD to depurinate the SRL, a similar approach could be explored for Stxs.
