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.

How Ricin Damages 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.

Functional Analysis of the Ribosomal uL6 Protein of Saccharomyces cerevisiae.

The genome-wide duplication event observed in eukaryotes represents an interesting biological phenomenon, extending the biological capacity of the genome at the expense of the same genetic material. For example, most ribosomal proteins in Saccharomyces cerevisiae are encoded by a pair of paralogous genes. It is thought that gene duplication may contribute to heterogeneity of the translational machinery; however, the exact biological function of this event has not been clarified. In this study, we have investigated the functional impact of one of the duplicated ribosomal proteins, uL6, on the translational apparatus together with its consequences for aging of yeast cells. Our data show that uL6 is not required for cell survival, although lack of this protein decreases the rate of growth and inhibits budding. The uL6 protein is critical for the efficient assembly of the ribosome 60S subunit, and the two uL6 isoforms most likely serve the same function, playing an important role in the adaptation of translational machinery performance to the metabolic needs of the cell. The deletion of a single uL6 gene significantly extends the lifespan but only in cells with a high metabolic rate. We conclude that the maintenance of two copies of the uL6 gene enables the cell to cope with the high demands for effective ribosome synthesis.

Structural properties of the human acidic ribosomal P proteins forming the P1-P2 heterocomplex.

The ribosome has a morphologically distinct structural feature called the stalk, recognized as a vital element for its function. The ribosomal P proteins constitute the main part of the eukaryotic ribosomal stalk, forming a pentameric structure P0-(P1-P2)(2). The group of P1/P2 proteins in eukaryotes is very diverse, and in spite of functional and structural similarities they do not fully complement one another, probably constituting an adaptive feature of the ribosome from a particular species to diverse environmental conditions. The functional differences among the P1/P2 proteins were analysed in vivo several times; however, a thorough molecular characterization was only done for the yeast P1/P2 proteins. Here, we report a biophysical analysis of the human P1 and P2 proteins, applying mass spectrometry, CD and fluorescence spectroscopy, cross-linking and size exclusion chromatography. The human P1/P2 proteins form stable heterodimer, as it is the case for P1/P2 from yeast. However, unlike the yeast complex P1A-P2B, the human P1-P2 dimer showed a three-state transition mechanism, suggesting that an intermediate species may exist in solution.

Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus.

The eukaryotic P-stalk contains two P1-P2 protein dimers with a conserved C- terminal domain (CTD) critical for the interaction with external factors. To understand the role of the individual CTD of human P1/P2 proteins, we examined the interaction of reconstituted human P-protein complexes and C-terminally truncated forms with ricin A chain (RTA), which binds to the stalk to depurinate the sarcin/ricin loop (SRL). The interaction between P-protein complexes and RTA was examined by surface plasmon resonance, isothermal titration calorimetry, microscale thermophoresis and bio-layer interferometry. The P1-P2 heterodimer missing a CTD on P2 was able to bind RTA. In contrast, the P1-P2 heterodimer missing the CTD of P1 protein displayed almost no binding toward RTA. Very low interaction was detected between RTA and the non-truncated P2-P2 homodimer, suggesting that the structural architecture of the P1-P2 heterodimer is critical for binding RTA. The reconstituted pentameric human stalk complex had higher affinity for RTA than the P1-P2 dimer. Deletion of P1 CTD, but not P2 CTD reduced the affinity of the pentamer for RTA. These results highlight the importance of the heterodimeric organization of P1-P2 in the human stalk pentamer and functional non-equivalence of the individual P-protein CTDs in the interaction with RTA.

SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY.

Frataxin is a highly conserved protein found in both prokaryotes and eukaryotes. It is involved in several central functions in cells, which include iron delivery to biochemical processes, such as heme synthesis, assembly of iron-sulfur clusters (ISC), storage of surplus iron in conditions of iron overload, and repair of ISC in aconitase. Frataxin from different organisms has been shown to undergo iron-dependent oligomerization. At least two different classes of oligomers, with different modes of oligomer packing and stabilization, have been identified. Here, we continue our efforts to explore the factors that control the oligomerization of frataxin from different organisms, and focus on E. coli frataxin CyaY. Using small-angle X-ray scattering (SAXS), we show that higher iron-to-protein ratios lead to larger oligomeric species, and that oligomerization proceeds in a linear fashion as a results of iron oxidation. Native mass spectrometry and online size-exclusion chromatography combined with SAXS show that a dimer is the most common form of CyaY in the presence of iron at atmospheric conditions. Modeling of the dimer using the SAXS data confirms the earlier proposed head-to-tail packing arrangement of monomers. This packing mode brings several conserved acidic residues into close proximity to each other, creating an environment for metal ion binding and possibly even mineralization. Together with negative-stain electron microscopy, the experiments also show that trimers, tetramers, pentamers, and presumably higher-order oligomers may exist in solution. Nano-differential scanning fluorimetry shows that the oligomers have limited stability and may easily dissociate at elevated temperatures. The factors affecting the possible oligomerization mode are discussed.

Multiplication of Ribosomal P-Stalk Proteins Contributes to the Fidelity of Translation.

The P-stalk represents a vital element within the ribosomal GTPase-associated center, which represents a landing platform for translational GTPases. The eukaryotic P-stalk exists as a uL10-(P1-P2)2 pentameric complex, which contains five identical C-terminal domains, one within each protein, and the presence of only one such element is sufficient to stimulate factor-dependent GTP hydrolysis in vitro and to sustain cell viability. The functional contribution of the P-stalk to the performance of the translational machinery in vivo, especially the role of P-protein multiplication, has never been explored. Here, we show that ribosomes depleted of P1/P2 proteins exhibit reduced translation fidelity at elongation and termination steps. The elevated rate of the decoding error is inversely correlated with the number of the P-proteins present on the ribosome. Unexpectedly, the lack of P1/P2 has little effect in vivo on the efficiency of other translational GTPase (trGTPase)-dependent steps of protein synthesis, including translocation. We have shown that loss of accuracy of decoding caused by P1/P2 depletion is the major cause of translation slowdown, which in turn affects the metabolic fitness of the yeast cell. We postulate that the multiplication of P-proteins is functionally coupled with the qualitative aspect of ribosome action, i.e., the recoding phenomenon shaping the cellular proteome.

The P1/P2 proteins of the human ribosomal stalk are required for ribosome binding and depurination by ricin in human cells.

Ricin A-chain (RTA) depurinates the sarcin-ricin loop of 28S ribosomal RNA and inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction of RTA with 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 of P1 and P2 bound to P0, whereas the yeast stalk consists of two different heterodimers, P1α-P2ß and P2α-P1ß, bound to P0. RTA exhibits 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 of RTA with human ribosomes and subsequent depurination of the sarcin-ricin loop. Using small interfering RNA-mediated knockdown of P1/P2 expression in human cells, we demonstrated that the depurination activity of RTA is lower when P1 and P2 levels are reduced. Biacore analysis showed that ribosomes from P1/P2-depleted cells have a reduced ability to bind RTA, which correlates with reduced depurination activity both in vitro and inside cells. RTA interacts directly with recombinant human P1-P2 dimer, further demonstrating the importance of human P1 and P2 in enabling RTA to bind and depurinate human ribosomes.

Subcellular localization of ribosomal P0-like protein MRT4 is determined by its N-terminal domain.

The Mrt4 protein, showing extensive sequence similarity to the ribosomal P0 protein, is classified as a ribosomal P0-like protein and acts as a trans-acting factor which modulates the assembly of the pre-60S particle. In this report we investigated the biological nature of the human Mrt4 protein. First, we constructed a series of hybrid hMrt4-P0 proteins by replacing various domains of the P0 protein with corresponding protein fragments from hMrt4. We found that hMrt4 binds to the same site on the large ribosomal subunit as does P0, but despite the sequence homology it is not able to functionally complement the lack of P0. Using fluorescence microscopy and biochemical approaches we also show that hMrt4 occupies predominantly the nucleolar compartment, in contrast to P0 and P1/P2, which are located in the cytoplasm. The nucleolar accumulation of hMrt4 does not depend on a specific nucleolus localization signal, but rather occurs via interaction with established nucleolar components such as rRNA; however, nuclear import of hMrt4 is dependent on a short sequence in the N-terminal part of the protein. Functional analysis with specific inhibitors, actinomycin D and leptomycin B, showed that hMrt4 is a trans-acting factor involved in ribosome maturation, with nucleus-cytoplasm shuttling capacity.

Structural relationships among the ribosomal stalk proteins from the three domains of life.

The GTPase center of the large ribosomal subunit, being a landing platform for translation factors, and regarded as one of the oldest structures in the ribosome, is a universally conserved structure in all domains of life. It is thought that this structure could be responsible for the major breakthrough on the way to the RNA/protein world, because its appearance would have dramatically increased the rate and accuracy of protein synthesis. The major part of this center is recognized as a distinct structural entity, called the stalk. The main functional part of the stalk in all domains of life is composed of small L12/P proteins, which are believed to form an evolutionarily conserved group. However, some data indicate that the bacterial and archaeo/eukaryal proteins are not related to each other structurally, and only a functional relationship may be clearly recognized. To clarify this point, we performed a comprehensive comparative analysis of the L12/P proteins from the three domains of life. The results show that bacterial and archaeo/eukaryal L12/P-proteins are not structurally related and, therefore, might not be linked evolutionarily either. Consequently, these proteins should be regarded as analogous rather than homologous systems and probably appeared on the ribosomal particle in two independent events in the course of evolution.