Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the ¹⁵N relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.

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.

Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their K_m values and catalytic rates (k_cat) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in K_m and up to 38-fold decrease in k_cat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.

RicinA‐chain (RTA) depurinates the sarcin–ricin loop of 28S ribosomalRNAand inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction ofRTAwith the ribosome and subsequent depurination. Despite homology between the stalk structures from yeast and humans, there are notable differences. The human ribosomal stalk contains two identical heterodimers ofP1 andP2 bound toP0, whereas the yeast stalk consists of two different heterodimers,P1α–P2β andP2α–P1β, bound toP0.RTAexhibits higher activity towards mammalian ribosomes than towards ribosomes from other organisms, suggesting that the mode of interaction with ribosomes may vary. Here, we examined whether the human ribosomal stalk proteins facilitate the interaction ofRTAwith human ribosomes and subsequent depurination of the sarcin–ricin loop. Using small interferingRNA‐mediated knockdown ofP1/P2 expression in human cells, we demonstrated that the depurination activity ofRTAis lower whenP1 andP2 levels are reduced. Biacore analysis showed that ribosomes fromP1/P2‐depleted cells have a reduced ability to bindRTA, which correlates with reduced depurination activity bothin vitroand inside cells.RTAinteracts directly with recombinant humanP1–P2 dimer, further demonstrating the importance of humanP1 andP2 in enablingRTAto bind and depurinate human ribosomes.Structured digital P2, P1 and RTA physically interact by surface plasmon resonance (View interaction) P2, P1 and RTA physically interact by anti bait coimmunoprecipitation (View interaction)