*2.1. Ricin Structure*

The first information about the bi-chain nature of ricin structure dates to the early 1970s, when it was shown that ricin was composed of two chains, A (active) and B (binding), linked together through a disulphide bond [26,27]. In the same period, the complete primary sequence of the ricin A and B chains was determined [28,29]. Ricin holotoxin structure was solved for the first time at 2.8 Å resolution (Figure 1) [30]. This pioneering work demonstrated that ricin A chain was a globular protein folded into three domains all contributing to the active site, while the B chain lectin folded into two domains, each binding lactose in a shallow cleft. The interface between the A and B chains showed some hydrophobic contact in which proline and phenylalanine side chains played a prominent role. Four years later, the same researchers refined ricin structure at 2.5 Å (Figure 2a), allowing a more detailed molecular description of the holotoxin and of the separated A and B chains [31–33]. Ricin A chain has been described as a globular protein consisting of 267 amino acids and organized in 8 α-helices and 8 β-strand structures. Ricin B chain consists of 262 amino acids and two homologues domains, each containing a lactose binding site and several areas of amino acid homology, possibly derived from a gene duplication. In 1995, after purification of a complex of ricin A chain cross-linked to the ribosome, it was found the binding of ricin A chain with the ribosomal proteins L9 and L10e [34,35].

**Figure 2.** (**a**) Ribbon model of the crystal structure of ricin at 2.5 Å (accession number Protein Data Bank 2AAI). The A chain domains are colored in green, blue, and light blue; the B chain domains are colored in yellow and orange. (**b**) Catalytic site of ricin. The key residues are indicated and colored in blue, whereas adenine substrate is depicted in red. (**c**) Proposed mechanism of depurination reaction catalyzed by ricin. The hydrolysis proceeds through a dissociative mechanism forming an oxocarbenium transition state. Arg180 protonates the leaving group and the N-glycosidic bond is broken. Glu177 deprotonates the hydrolytic water (highlighted by a red dotted rectangle) that attacks carbon to complete the depurination reaction. Figure 2a and 2b were produced by PyMOL (version 2.3.1); Figure 2c was produced by ACD/ChemSketch (version 2015.2.5).

The knowledge of the tridimensional structure of ricin yielded more information on its active site. Studies based on the formation of complexes between the A chain, both native and recombinant, and adenine-containing nucleotides allowed for the identification of key residues in enzymatic activity. In particular, Tyr80, Tyr123, Glu177, Arg180, and Trp211 were found to form the binding site for adenine (Figure 2b) [30,36]. In the 1990s, the molecular mechanism of adenine release was hypothesized: Adenine is sandwiched between Tyr80 and Tyr123 in a π stacking interaction; the N3 of adenine is protonated by Arg180, promoting the C1'-N9 bond breaking, and thus forming an oxocarbenium moiety on the ribose (Figure 2c) [36,37]. This transition state is stabilized by Glu177; a water molecule lies on the opposite side of the sugar ring from adenosine, which will be polarized by Arg180 to a hydroxide character that rapidly attacks the sugar carbon completing the reaction.
