Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inactivates ribosome by enzymatically depurinating the A⁴³²⁴ at the α-sarcin/ricin loop of 28S rRNA. We have shown in this and previous studies that TCS interacts with human acidic ribosomal proteins P0, P1 and P2, which constitute the lateral stalk of eukaryotic ribosome. Deletion mutagenesis showed that TCS interacts with the C-terminal tail of P2, the sequences of which are conserved in P0, P1 and P2. The P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation experiments. Scanning charge-to-alanine mutagenesis has shown that K173, R174 and K177 in the C-terminal domain of TCS are involved in interacting with the P2, presumably through forming charge–charge interactions to the conserved DDD motif at the C-terminal tail of P2. A triple-alanine variant K173A/R174A/K177A of TCS, which fails to bind P2 and ribosomal stalk in vitro, was found to be 18-fold less active in inhibiting translation in rabbit reticulocyte lysate, suggesting that interaction with P-proteins is required for full activity of TCS. In an analogy to the role of stalk proteins in binding elongation factors, we propose that interaction with acidic ribosomal stalk proteins help TCS to locate its RNA substrate.     

Structure of a Two-Domain N-Terminal Fragment of Ribosomal Protein L10 from Methanococcus jannaschii Reveals a Specific Piece of the Archaeal Ribosomal Stalk

Ribosomal stalk is involved in the formation of the so-called “GTPase-associated site” and plays a key role in the interaction of ribosome with translation factors and in the control of translation accuracy. The stalk is formed by two or three copies of the L7/L12 dimer bound to the C-terminal tail of protein L10. The N-terminal domain of L10 binds to a segment of domain II of 23S rRNA near the binding site for ribosomal protein L11. The structure of bacterial L10 in complex with three L7/L12 N-terminal dimers has been determined in the isolated state, and the structure of the first third of archaeal L10 bound to domain II of 23S rRNA has been solved within the Haloarcula marismortui 50S ribosomal subunit. A close structural similarity between the RNA-binding domain of archaeal L10 and the RNA-binding domain of bacterial L10 has been demonstrated. In this work, a long RNA-binding N-terminal fragment of L10 from Methanococcus jannaschii has been isolated and crystallized. The crystal structure of this fragment (which encompasses two-thirds of the protein) has been solved at 1.6 Å resolution. The model presented shows the structure of the RNA-binding domain and the structure of the adjacent domain that exist in archaeal L10 and eukaryotic P0 proteins only. Furthermore, our model incorporated into the structure of the H. marismortui 50S ribosomal subunit allows clarification of the structure of the archaeal ribosomal stalk base.     

The acidic protein binding site is partially hidden in the free Saccharomyces cerevisiae ribosomal stalk protein P0

The ribosomal stalk is essential for translation; however, its overall structure is poorly understood. Characterization of the region involved in the interactions between protein P0 and the 12 kDa acidic proteins P1 and P2 is fundamental to understand the assembly and function of this structure in the eukaryotic ribosome. The acidic protein content is important for the ribosome efficiency and affects the translation of specific mRNAs. By usage of a series of progressively truncated fragments of protein P0 in the two-hybrid test, a region between positions 213 and 250 was identified as the minimal protein part able to interact with the acidic proteins. Extensions at either end affect the binding capacity of the fragment either positively or negatively depending on the number of added amino acids. Deletions inside the binding region confirm its in vivo relevance since they drastically reduce the P0 interacting capacity with the 12 kDa acidic proteins, which are severely reduced in the ribosome when the truncated protein is expressed in the cell. Moreover, recombinant His-tagged P0 fragments containing the binding site and bound to Ni(2+)-NTA columns can form a complex with the P1 and P2 proteins, which is able to bind elongation factor EF2. The results indicate the existence of a region in P0 that specifically interacts with the acidic proteins. These interactions are, however, hindered by the presence of neighbor protein domains, suggesting the need for conformational changes in the complete P0 to allow the assembly of the ribosomal stalk.     

Structural and functional characterization of the amino terminal domain of the yeast ribosomal stalk P1 and P2 proteins

The essential ribosomal stalk is formed in eukaryotes by a pentamer of two P1–P2 protein heterodimers and the P0 rRNA binding protein. In contrast to the highly stable prokaryotic complex, the P1 and P2 proteins in the eukaryotic stalk undergo a cyclic process of assembly and disassembly during translation that seems to modulate the ribosome activity. To better understand this process, the regions of the Saccharomyces cerevisiae P1α and P2β proteins that are directly involved in heterodimer formation and ribosome binding have been characterized using a series of P1α/P2β chimeras. The region required for a stable interaction with the ribosome is formed by the first three predicted α-helices in the N-terminal domain of both proteins. The same region is required for heterodimer formation in P2β but the third helix is dispensable for this association in P1α. It seems, therefore, that stable ribosome binding is more structurally demanding than heterodimerization. A fourth predicted α-helix in the N-terminal domain of P1α and P2β appears not to be involved in the assembly process but rather, it contributes to the conformation of the proteins by apparently restricting the mobility of their C-terminal domain and paradoxically, by reducing their activity. In addition, the study of P1/P2 chimeras showed that the C-terminal domains of these two types of protein are functionally identical and that their protein specificity is exclusively determined by their N-terminal domains.     

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.     

Structural Basis for Translation Factor Recruitment to the Eukaryotic/Archaeal Ribosomes

The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with eukaryotic pentameric stalk complex; it provides access to eukaryotic elongation factors at levels comparable to that of the eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)₂(P1)₂(P1)₂) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.     

A Novel Association between Two Trypanosome-Specific Factors and the Conserved L5-5S rRNA Complex

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are involved in and essential for ribosome biogenesis. The proteins interact with the 5S rRNA with nearly identical binding characteristics. We have shown that this interaction is achieved mainly through the LoopA region of the RNA, but P34 and P37 also protect the L5 binding site located on LoopC. We now provide evidence to show that these factors form a novel pre-ribosomal particle through interactions with both 5S rRNA and the L5 ribosomal protein. Further in silico and in vitro analysis of T. brucei L5 indicates a lower affinity for 5S rRNA than expected, based on other eukaryotic L5 proteins. We hypothesize that P34 and P37 complement L5 and bridge the interaction with 5S rRNA, stabilizing it and aiding in the early steps of ribosome biogenesis.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.     

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2

The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF‐2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1β, TcP2α, TcP2β and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two‐hybrid (Y2H) protein–protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1β–TcP2α and TcP1β–TcP2β. Moreover P2 but not P1 proteins were able to homo‐oligomerize. In addition, the region comprising amino acids 210–270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2β were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF‐2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF‐2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0. Copyright © 2010 John Wiley & Sons, Ltd.     

The amino terminal end determines the stability and assembling capacity of eukaryotic ribosomal stalk proteins P1 and P2

The eukaryotic ribosomal proteins P1 and P2 bind to protein P0 through their N-terminal domain to form the essential ribosomal stalk. A mutational analysis points to amino acids at positions 2 and 3 as determinants for the drastic difference of Saccharomyces cerevisiae P1 and P2 half-life, and suggest different degradation mechanisms for each protein type. Moreover, the capacity to form P1/P2 heterodimers is drastically affected by mutations in the P2β four initial amino acids, while these mutations have no effect on P1β. Binding of P2β and, to a lesser extent, P1β to the ribosome is also seriously affected showing the high relevance of the amino acids in the first turn of the NTD α-helix 1 for the stalk assembly. The negative effect of some mutations on ribosome binding can be reversed by the presence of the second P1/P2 couple in the ribosome, indicating a stabilizing structural influence between the two heterodimers. Unexpectedly, some mutations totally abolish heterodimer formation but allow significant ribosome binding and, therefore, a previous P1 and P2 association seems not to be an absolute requirement for stalk assembly. Homology modeling of the protein complexes suggests that the mutated residues can affect the overall protein conformation.     

Solution structure of the dimerization domain of ribosomal protein P2 provides insights for the structural organization of eukaryotic stalk

The lateral stalk of ribosome is responsible for kingdom-specific binding of translation factors and activation of GTP hydrolysis that drives protein synthesis. In eukaryotes, the stalk is composed of acidic ribosomal proteins P0, P1 and P2 that constitute a pentameric P-complex in 1: 2: 2 ratio. We have determined the solution structure of the N-terminal dimerization domain of human P2 (NTD-P2), which provides insights into the structural organization of the eukaryotic stalk. Our structure revealed that eukaryotic stalk protein P2 forms a symmetric homodimer in solution, and is structurally distinct from the bacterial counterpart L12 homodimer. The two subunits of NTD-P2 form extensive hydrophobic interactions in the dimeric interface that buries 2400 Ų of solvent accessible surface area. We have showed that P1 can dissociate P2 homodimer spontaneously to form a more stable P1/P2 1 : 1 heterodimer. By homology modelling, we identified three exposed polar residues on helix-3 of P2 are substituted by conserved hydrophobic residues in P1. Confirmed by mutagenesis, we showed that these residues on helix-3 of P1 are not involved in the dimerization of P1/P2, but instead play a vital role in anchoring P1/P2 heterodimer to P0. Based on our results, models of the eukaryotic stalk complex were proposed.     

Archaeal ribosomal protein L1: the structure provides new insights into RNA binding of the L1 protein family

BACKGROUND: L1 is an important primary rRNA-binding protein, as well as a translational repressor that binds mRNA. It was shown that L1 proteins from some bacteria and archaea are functionally interchangeable within the ribosome and in the repression of translation. The crystal structure of bacterial L1 from Thermus thermophilus (TthL1) has previously been determined. RESULTS: We report here the first structure of a ribosomal protein from archaea, L1 from Methanococcus jannaschii (MjaL1). The overall shape of the two-domain molecule differs dramatically from that of its bacterial counterpart (TthL1) because of the different relative orientations of the domains. Two strictly conserved regions of the amino acid sequence, each belonging to one of the domains and positioned close to each other in the interdomain cavity of TthL1, are separated by about 25 A in MjaL1 owing to a significant opening of the structure. These regions are structurally highly conserved and are proposed to be the specific RNA-binding sites. CONCLUSIONS: The unusually high RNA-binding affinity of MjaL1 might be explained by the exposure of its highly conserved regions. The open conformation of MjaL1 is strongly stabilized by nonconserved interdomain interactions and suggests that the closed conformations of L1 (as in TthL1) open upon RNA binding. Comparison of the two L1 protein structures reveals a high conformational variability of this ribosomal protein. Determination of the MjaL1 structure offers an additional variant for fitting the L1 protein into electron-density maps of the 50S ribosomal subunit.     

RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1

The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation. Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein. We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit. The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay. CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity. Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C). In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5′-untranslated region of the mRNA in comparison to the other parts. RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein. These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5′-untranslated region. In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine–Dalagarno sequences. In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.     

Phosphorylation of the yeast ribosomal stalk. Functional effects and enzymes involved in the process

The ribosomal stalk is directly involved in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes the acidic components correspond to the 12-kDa P1 and P2 proteins, and the RNA binding component is the P0 protein. All these proteins are found phosphorylated in eukaryotic organisms, and previous in vitro data suggested this modification was involved in the activity of this structure. Results from mutational studies have shown that phosphorylation takes place at a serine residue close to the carboxy end of the P proteins. Modification of this serine residue does not affect the formation of the stalk and the activity of the ribosome in standard conditions but induces an osmoregulation-related phenotype at 37 degrees C. The phosphorylatable serine is part of a consensus casein kinase II phosphorylation site. However, although CKII seems to be responsible for part of the stalk phosphorylation in vivo, it is probably not the only enzyme in the cell able to perform this modification. Five protein kinases, RAPI, RAPII and RAPIII, in addition to the previously reported CKII and PK60 kinases, are able to phosphorylate the stalk proteins. A comparison of the five enzymes shows differences among them that suggest some specificity regarding the phosphorylation of the four yeast acidic proteins. It has been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data suggest that although phosphorylation is not involved in the interaction of the acidic P proteins with the ribosome, it can affect the ribosome activity and might participate in a possible ribosome regulatory mechanism.     

The subcellular distribution of the human ribosomal "stalk" components: P1, P2 and P0 proteins

The ribosomal "stalk" structure is a distinct lateral protuberance located on the large ribosomal subunit in prokaryotic, as well as in eukaryotic cells. In eukaryotes, this ribosomal structure is composed of the acidic ribosomal P proteins, forming two hetero-dimers (P1/P2) attached to the ribosome through the P0 protein. The "stalk" is essential for the ribosome activity, taking part in the interaction with elongation factors.In this report, we have shown that the subcellular distribution of the human P proteins does not fall into standard behavior of regular ribosomal proteins. We have used two approaches to assess the distribution of the P proteins, in vivo experiments with GFP fusion proteins and in vitro one with anti-P protein antibodies. In contrast to standard r-proteins, the P1 and P2 proteins are not actively transported into the nucleus compartment, remaining predominantly in the cytoplasm (the perinuclear compartment). The P0 protein was found in the cytoplasm, as well as in the nucleus; however, the nucleoli were excluded. This protein was scattered around the nuclei, and the distribution might reflect association with the so-called nuclear bodies. This is the first example of r-proteins that are not actively transported into the nucleus; moreover, this might imply that the "stalk" constituents are assembled onto the ribosomal particle at the very last step of ribosomal maturation, which takes part in the cell cytoplasm.     

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.