Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk reveals residues involved in the binding interaction.

Shiga toxin 2a (Stx2a) is the virulence factor of enterohemorrhagic Escherichia coli. The catalytic A1 subunit of Stx2a (Stx2A1) interacts with the ribosomal P-stalk for loading onto the ribosome and depurination of the sarcin-ricin loop, which halts protein synthesis. Because of the intrinsic flexibility of the P-stalk, a structure of the Stx2a-P-stalk complex is currently unknown. We demonstrated that the native P-stalk pentamer binds to Stx2a with nanomolar affinity, and we employed cryo-EM to determine a structure of the 72 kDa Stx2a complexed with the P-stalk. The structure identifies Stx2A1 residues involved in binding and reveals that Stx2a is anchored to the P-stalk via only the last six amino acids from the C-terminal domain of a single P-protein. For the first time, the cryo-EM structure shows the loop connecting Stx2A1 and Stx2A2, which is critical for activation of the toxin. Our principal component analysis of the cryo-EM data reveals the intrinsic dynamics of the Stx2a-P-stalk interaction, including conformational changes in the P-stalk binding site occurring upon complex formation. Our computational analysis unveils the propensity for structural rearrangements within the C-terminal domain, with its C-terminal six amino acids transitioning from a random coil to an α-helix upon binding to Stx2a. In conclusion, our cryo-EM structure sheds new light into the dynamics of the Stx2a-P-stalk interaction and indicates that the binding interface between Stx2a and the P-stalk is the potential target for drug discovery.

Functional divergence between the two P1-P2 stalk dimers on the ribosome in their interaction with ricin A chain.

The eukaryotic stalk, which is responsible for the recruitment of translation factors, is a pentamer containing two P1-P2 dimers with unclear modes of action. In Saccharomyces cerevisiae, P1/P2 proteins (individual P1 and P2 proteins) are organized into two distinct dimers, P1A-P2B and P1B-P2A. To investigate the functional contribution of each dimer on the ribosome, RTA (ricin A chain), which binds to the stalk to depurinate the SRL (sarcin/ricin loop), was used as a molecular probe in yeast mutants in which the binding site for one or the other dimer on P0 was deleted. Ribosome depurination and toxicity of RTA were greatly reduced in mutants containing only P1A-P2B on the ribosome, whereas those with only P1B-P2A were reduced less in depurination and were unaffected in toxicity. Ribosomes bearing P1B-P2A were depurinated by RTA at a similar level as wild-type, but ribosomes bearing P1A-P2B were depurinated at a much lower level in vitro. The latter ribosomes showed the lowest association and almost no dissociation with RTA by surface plasmon resonance. These results indicate that the P1B-P2A dimer is more critical for facilitating the access of RTA to the SRL, providing the first in vivo evidence for functional divergence between the two stalk dimers on the ribosome.

Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk.

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 kcat 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.

Solution structure of the natively assembled yeast ribosomal stalk determined by small-angle X-ray scattering.

The ribosomal stalk of the 60S subunit has been shown to play a crucial role in all steps of protein synthesis, but its structure and exact molecular function remain an unanswered question. In the present study, we show the low-resolution models of the solution structure of the yeast ribosomal stalk, composed of five proteins, P0-(P1-P2)(2). The model of the pentameric stalk complex determined by small-angle X-ray scattering reveals an elongated shape with a maximum length of 13 nm. The model displays three distinct lobes, which may correspond to the individual P1-P2 heterodimers anchored to the C-terminal domain of the P0 protein.

Pentameric organization of the ribosomal stalk accelerates recruitment of ricin a chain to the ribosome for depurination.

Ribosome inactivating proteins (RIPs) depurinate a universally conserved adenine in the α-sarcin/ricin loop (SRL) and inhibit protein synthesis at the translation elongation step. We previously showed that ribosomal stalk is required for depurination of the SRL by ricin toxin A chain (RTA). The interaction between RTA and ribosomes was characterized by a two-step binding model, where the stalk structure could be considered as an important interacting element. Here, using purified yeast ribosomal stalk complexes assembled in vivo, we show a direct interaction between RTA and the isolated stalk complex. Detailed kinetic analysis of these interactions in real time using surface plasmon resonance (SPR) indicated that there is only one type of interaction between RTA and the ribosomal stalk, which represents one of the two binding steps of the interaction with ribosomes. Interactions of RTA with the isolated stalk were relatively insensitive to salt, indicating that nonelectrostatic interactions were dominant. We compared the interaction of RTA with the full pentameric stalk complex containing two pairs of P1/P2 proteins with its interaction with the trimeric stalk complexes containing only one pair of P1/P2 and found that the rate of association of RTA with the pentamer was higher than with either trimer. These results demonstrate that the stalk is the main landing platform for RTA on the ribosome and that pentameric organization of the stalk accelerates recruitment of RTA to the ribosome for depurination. Our results suggest that multiple copies of the stalk proteins might also increase the scavenging ability of the ribosome for the translational GTPases.

Cerrena unicolor Laccases, Genes Expression and Regulation of Activity.

A white rot fungus Cerrena unicolor has been identified as an important source of laccase, unfortunately regulation of this enzyme genes expression is poorly understood. Using 1D and 2D PAGE and LC-MS/MS, laccase isoenzymes were investigated in the liquid filtrate of C. unicolor culture. The level of expression of laccase genes was measured using qPCR. The elevated concentrations of copper and manganese in the medium caused greatest change in genes expression and three laccase transcripts were significantly affected after culture temperature was decreased from 28 to 4 °C or increased to 40 °C. The small differences in the PAGE band intensities of individual laccase proteins were also observed, indicating that given compound affect particular laccase's transcript. Analyses of laccase-specific activity, at all tested conditions, showed the increased activities as compared to the control, suggesting that enzyme is regulated at the post-translational stage. We observed that the aspartic protease purified from C. unicolor, significantly stimulate laccase activity. Moreover, electrochemical analysis of protease-treated laccase sample had 5 times higher redox peaks. The obtained results indicate that laccases released by C. unicolor are regulated at transcriptional, translational, and at the post-translational steps of gene expression helping fungus adapt to the environmental changes.

Phosphorylation of the conserved C-terminal domain of ribosomal P-proteins impairs the mode of interaction with plant toxins.

The ribosome is subjected to post-translational modifications, including phosphorylation, that affect its biological activity. Among ribosomal elements, the P-proteins undergo phosphorylation within the C terminus, the element which interacts with trGTPases or ribosome-inactivating proteins (RIPs); however, the role of phosphorylation has never been elucidated. Here, we probed the function of phosphorylation on the interaction of P-proteins with RIPs using the ribosomal P1-P2 dimer. We determined the kinetic parameters of the interaction with the toxins using biolayer interferometry and microscale thermophoresis. The results present the first mechanistic insight into the function of P-protein phosphorylation, showing that introduction of a negative charge into the C terminus of P1-P2 proteins promotes α-helix formation and decreases the affinity of the P-proteins for the RIPs.

Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation.

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 (15)N 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.

Biophysical properties of the eukaryotic ribosomal stalk.

The landing platform for the translational GTPases is located on the 60S ribosomal subunit and is referred to as a GTPase-associated center. The most distinctive feature of this center is an oligomeric complex, the stalk, responsible for the recruitment of translation factors and stimulation of translation factor-dependent GTP hydrolysis. In eukaryotes, the stalk has been investigated in vitro and in vivo, but most information available concerns its individual components only. In the present study, we provide an insight into the biophysical nature of the native stalk isolated from the yeast Saccharomyces cerevisiae. Using fluorescence, circular dichroism, and mass spectrometry analyses, we were able to characterize the natively formed yeast stalk, casting new light on the oligomeric properties of the complex and its quaternary topology, showing that folding and assembly are coupled processes. The pentameric stalk is an exceptionally stable structure with the protein core composed of P0, P1A, and P2B proteins and less tightly bound P1B and P2A capable of dissociating from the stalk core. We obtained also the whole picture of the posttranslational modifications at the logarithmic phase of yeast growth, using mass spectrometry approach, where P proteins are phosphorylated at a single serine residue, P0 may accept two phosphate groups, and P1A none. Additionally, only P1B undergoes N-terminal acetylation after prior methionine removal.

Ribosomal Protein uL11 as a Regulator of Metabolic Circuits Related to Aging and Cell Cycle.

Aging is a biological phenomenon common to all living organisms. It is thought that the rate of aging is influenced by diverse factors, in many cases related to the control of energy metabolism, i.e., the so-called pro-longevity effects of starvation. Translation, regarded as the main energy consumption process, lies at the center of interest, as it has a significant impact on the longevity phenomenon. It has been shown that perturbations in the translational apparatus may lead to a lower rate of aging. Therefore, the main aim of this study was to investigate aging in relation to the protein biosynthesis circuit, taking into account the uL11 ribosomal protein as a vital ribosomal element. To this end, we used set of yeast mutants with deleted single uL11A or uL11B genes and a double disruptant uL11AB mutant. We applied an integrated approach analyzing a broad range of biological parameters of yeast mutant cells, especially the longevity phenomenon, supplemented with biochemical and high throughput transcriptomic and metobolomic approaches. The analysis showed that the longevity phenomenon is not fully related to the commonly considered energy restriction effect, thus the slow-down of translation does not represent the sole source of aging. Additionally, we showed that uL11 can be classified as a moonlighting protein with extra-ribosomal function having cell-cycle regulatory potential.