How Ricin Damages the Ribosome

Abstract

Ricin belongs to the group of ribosome-inactivating proteins (RIPs), i.e., toxins that have evolved to provide particular species with an advantage over other competitors in nature. Ricin possesses RNA N-glycosidase activity enabling the toxin to eliminate a single adenine base from the sarcin-ricin RNA loop (SRL), which is a highly conserved structure present on the large ribosomal subunit in all species from the three domains of life. The SRL belongs to the GTPase associated center (GAC), i.e., a ribosomal element involved in conferring unidirectional trajectory for the translational apparatus at the expense of GTP hydrolysis by translational GTPases (trGTPases). The SRL represents a critical element in the GAC, being the main triggering factor of GTP hydrolysis by trGTPases. Enzymatic removal of a single adenine base at the tip of SRL by ricin blocks GTP hydrolysis and, at the same time, impedes functioning of the translational machinery. Here, we discuss the consequences of SRL depurination by ricin for ribosomal performance, with emphasis on the mechanistic model overview of the SRL modus operandi.

Keywords: GTPase associated center (GAC); P-proteins; ribosome; ribosome-inactivating protein; ricin; sarcin-ricin loop (SRL); translation.

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 yeast 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).

Figure 2

The model of sarcin–ricin loop structure. (A) Alignment of highly conserved secondary structures of yeast and E. coli SRL. The red color indicates the key adenine hydrolyzed by the ricin. A conserved fragment of 12 nucleotides is marked with a gray color. (B) Structure of the S. cerevisiae SRL (PDB code 3U5H). The key adenine was marked in red (A₃₀₂₇). The individual structural elements of the SRL - the stem, the flexible region, the G-bulged cross-strand stack with the highlighted individual nucleotides and the GAGA loop - are marked with a dotted black line. The gray fields show the non-canonical π-stacking interactions between particular bases. Model prepared by PyMol software, (The PyMOL Molecular Graphics System, version 1.5.0.4, Schrödinger, LLC, NY, USA) based on [42].

Figure 3

Model of GTP hydrolysis activation with the aid of sarcin–ricin loop (SRL), with the EF-G as trGTPase. Left panel: the organization of the active site in isolated EF-G. Asp₂₂ and His₈₇ are in “flipped-out” state, pointing away from GTP; the “hydrophobic gate” formed by amino acids Ile₆₃ and Ile₂₁ prevents His₈₇ from adopting the active conformation. Right panel: the reorganization of the active site of EF-G as a result of binding of EF-G to ribosome and inserting the SRL to the G-domain. Base A₂₆₆₀ interacts with His₂₀, which induces Ile₂₁ movement away from GTP and “hydrophobic gate” opening. The phosphate of A₂₆₆₂ directs His₈₇ and Asp₂₂ residues (through Mg²⁺) to “flipped-in” conformation which allows for water molecule activation and GTP hydrolysis (see the details in the text).

Figure 4

Model of sarcin–ricin loop (SRL) depurination by RTA. Removal of key A₂₆₆₀ residue (E. coli numbering) impairs the intricate interaction network responsible for stabilization of the active state of trGTPases, resulting in the inhibition of translation process. The base of A₂₆₆₀ is bound to the catalytic center of RTA with π-stacking interactions and its position is stabilized by hydrogen bonds with Gly₁₂₁, Val₈₁ and Arg₁₈₀ (green). Glu₁₇₇ and Arg₁₈₀ (green) play crucial role in catalysis by transition state stabilization. Arg₁₈₀ residue protonates base of A₂₆₆₀ causing delocalization of ring electrons. Glu₁₇₇ polarizes water molecule (blue) and resultant hydroxide ion attacks positive center on ribose which leads to hydrolysis of N-glycosidic bond; prepared based on [110].