**1. Introduction**

Toxic proteins are naturally present in a wide variety of species [1]. It is thought that they have evolved to play a specific role in defense against animals, pathogens, and various insects, giving advantage in a particular niche [2,3]. One of the most toxic proteins known in nature are plant toxins from the class of ribosome-inactivating proteins. The mechanism of toxicity of plant toxins is of grea<sup>t</sup> interest because they are present in human foods [4,5] and used in ethnomedicine [6] and cosmetics [7]. Currently, they have attracted attention in broad biotechnological applications [8,9]. Importantly, they also pose a significant threat, as numerous plant toxins, such as ricin, have been recognized as a biological weapon [10] and are recently considered as an agen<sup>t</sup> of bioterrorism [11]. Ricin is one of the most common and potent lethal biological molecules known [9]. Ricin and related proteins display one common feature, i.e. the ability to inhibit ribosomes, and this particular feature laid the foundation for the general name for all these proteins: "ribosome-inactivating proteins" (RIPs) [12]. In general, all RIPs have been classified as single chain (type 1) and double chain (type 2) proteins [13]. Type 1 RIPs consist of an enzymatically active chain (A) and include for instance: pokeweed antiviral protein (PAP), trichosanthin, saporin, gelonin, and lu ffin. Ricin, abrin, and Shiga toxin are classified as type 2 RIPs, where the enzymatically active A-chain is disulfide-linked to a B-chain, which provides an active chain with higher ability to enter cells. Thus, type 2 RIPs are considerably more toxic than type 1 RIPs [13]. Also, a third class of RIPs, termed type 3, has been recognized. It includes only a few members, with the prominent example of jasmonate induced protein (JIP60) [14] and maize b-32 protein, which requires proteolytic process to become active [15]. In 2010, a new nomenclature has been proposed for RIPs, in which they are termed A (type 1), AB (type 2), AC (type 3, similar to JIP60), and AD (Type 3, similar to b32) [16]. The current model of the catalytic activity of ricin and other RIPs assumes that, using the N-glycosidase activity, the toxin removes a single adenine base from the ribosomal RNA (rRNA) located at the heart of the ribosomal GTPase associated center (GAC) recognized as a central ribosomal element responsible for fueling of the translational machinery [17]. The prime target of ricin is a structural element of rRNA and is called the sarcin–ricin loop (SRL) because this element is targeted by another ribo-toxin, α-sarcin; ye<sup>t</sup> in this case, the SRL undergoes endonucleolytic cleavage [18]. The ricin-dependent depurination of the SRL exerts a deleterious effect, blocking the ribosome action and thereby hampering protein synthesis. It should be stressed that despite the high homology of the SRL within the three domains of life, mainly eukaryotic ribosomes undergo efficient depurination, however, several type I RIPs were shown to depurinate prokaryotic ribosomes as well [19]. The extraordinary specificity of the RIP action is based on their interaction with unique eukaryotic proteins; in the case of ricin, the ribosomal P-proteins are regarded as a main docking part [20–22] while the L3 protein represents the main landing platform for the PAP [23,24]. However, regardless of the docking element, the eukaryotic ribosomal proteins are considered as guide molecules for RIPs, directing and activating the toxin toward its catalytic target - the SRL. The toxicity of ricin was mainly associated with inhibition of protein synthesis, but it should be stressed that there is no clear link between depurination of SRL, ribosome hampering and the toxic effect in the cell. The currently available information concerning ricin toxicity suggests that the toxic effect on cell metabolism is multifactorial and involves induction of numerous pathways that lead to cell death; however, molecular details elucidating the effect of ricin on cell metabolism are still elusive [25].

The numerous biochemical approaches [26–29] and especially current structural analyses have provided deep insight into the role of SRL in ribosome performance and cast light on the molecular consequences of its depurination-induced damage. Here, we are presenting the current understanding of the structure and function of SRL during the translational cycle, with particular focus on the molecular consequences of ricin-dependent adenine base removal on ribosome performance.

#### **2. Ribosome Structure as A Prime Target for Ricin**

#### *2.1. Ribosome and Its Active Sites*

Protein biosynthesis, being a critical biological pathway, provides a suitable target for many toxins or natural inhibitors with the ribosome as the main objective [30,31]. The ribosome is one of the most conserved and sophisticated macromolecular machines of the cell in all domains of life [32]. It is composed of a small and a large subunit, which together form a fully functional ribosome. Ribosomal subunits are composed of ribosomal RNA (rRNA) and a large number of ribosomal proteins, but it is the rRNA that plays the most critical functional role, defining the ribosome as a ribozyme [33]. The ribosome can be regarded as a scaffolding platform for many molecules, e.g., mRNA, aminoacyl-tRNAs (aa-tRNA), and protein factors, which together form the translational machinery converting genetic information into functional proteins [34]. The ribosome, being the central element of this machinery, carries out its task through several functional elements. The small subunit decodes the genetic information delivered by mRNA, whereas the large subunit hosts the catalytic peptidyl transferase center (PTC), where amino acids delivered by aa-tRNAs are linked into polypeptides [32]. Additionally, three tRNA binding sites can be recognized on the ribosome: aminoacyl (A), peptidyl (P), and exit (E) sites. The A site accommodates the incoming aa-tRNA, the P site binds the peptidyl-tRNA thus carrying the nascent polypeptide chain, and the E site binds deacylated tRNA before it dissociates from the ribosome. On the large ribosomal subunit there is also the GTPase associated center (GAC) which belongs to the ribosomal elements responsible for administrating the continuous motions of the ribosome along the translational cycle.

## 2.1.1. The GTPase Associated Center

The translation process to proceed efficiently and to comply with the metabolic needs of the cell requires many protein factors, which sequentially guide the ribosome through the protein synthesis cycle. The most critical group of factors are proteins that bind and hydrolyze GTP, called translational GTPases (trGTPases), which confer the unidirectional trajectory of the translational machinery at the expense of energy released from GTP hydrolysis [17]. The landing platform for all trGTPases is situated on the large ribosomal subunit and is called the GTPase associated center (GAC) (Figure 1) [35].

**Figure 1.** Model of GTPase associated center (GAC). Left panel: scheme of the 80S ribosome with the ribosomal P-stalk shown with extended C-terminal regions and the sarcin–ricin loop (SRL) (red). Right panel: fragment of 60S *S. cerevisiae* ribosome 25S rRNA (PDB code 3U5H) and 60S subunit (PDB code 3U5I); 25S rRNA and ribosomal proteins are indicated as light gray and dark gray colors, respectively. The fitted schematic structure of uL10 protein fragment in complex with the N-terminal domains of P1/P2-proteins (PDB code 3A1Y) from Archaea is depicted as dark blue and marine blue, respectively. The position of the yeas<sup>t</sup> 60S subunit is oriented to show A3027 of the SRL and positions of uL11 (slate blue), uL40 (deep blue) and uL6 (sky blue) proteins. Model prepared with PyMol software (The PyMOL Molecular Graphics System, version 1.5.0.4, Schrödinger, LLC, NY, USA).

The GAC is responsible for recruitment of trGTPases and stimulation of factor-dependent GTP hydrolysis [36]. The center consists of two main functional elements: a conserved fragment of rRNA called the sarcin–ricin loop (SRL) and the ribosomal stalk, composed exclusively of ribosomal proteins [37]. Both elements are critical for activation of trGTPases [28,38], and mutual cooperativity of the SRL and the stalk elements has been shown to be pivotal in stimulation of GTP hydrolysis by trGTPases [39–41].
