Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes.

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength.

Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3'end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region.

Insights into the Hendra virus NTAIL-XD complex: Evidence for a parallel organization of the helical MoRE at the XD surface stabilized by a combination of hydrophobic and polar interactions.

The Hendra virus is a member of the Henipavirus genus within the Paramyxoviridae family. The nucleoprotein, which consists of a structured core and of a C-terminal intrinsically disordered domain (N(TAIL)), encapsidates the viral genome within a helical nucleocapsid. N(TAIL) partly protrudes from the surface of the nucleocapsid being thus capable of interacting with the C-terminal X domain (XD) of the viral phosphoprotein. Interaction with XD implies a molecular recognition element (MoRE) that is located within N(TAIL) residues 470-490, and that undergoes α-helical folding. The MoRE has been proposed to be embedded in the hydrophobic groove delimited by helices α2 and α3 of XD, although experimental data could not discriminate between a parallel and an antiparallel orientation of the MoRE. Previous studies also showed that if the binding interface is enriched in hydrophobic residues, charged residues located close to the interface might play a role in complex formation. Here, we targeted for site directed mutagenesis two acidic and two basic residues within XD and N(TAIL). ITC studies showed that electrostatics plays a crucial role in complex formation and pointed a parallel orientation of the MoRE as more likely. Further support for a parallel orientation was afforded by SAXS studies that made use of two chimeric constructs in which XD and the MoRE were covalently linked to each other. Altogether, these studies unveiled the multiparametric nature of the interactions established within this complex and contribute to shed light onto the molecular features of protein interfaces involving intrinsically disordered regions.

Ubiquitin-independent proteasomal degradation.

Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide insights into the general requirements for proteasomal degradation. Here the focus is on three ubiquitin-independent substrates that have been the subject of detailed study. These are Rpn4, a transcriptional regulator of proteasome homeostasis, thymidylate synthase, an enzyme required for production of DNA precursors and ornithine decarboxylase, the initial enzyme committed to polyamine biosynthesis. It can be inferred from these cases that proteasome association and the presence of an unstructured region are the sole prerequisites for degradation. Based on that inference, artificial substrates have been designed to test the proteasome's capacity for substrate processing and its limitations. Ubiquitin-independent substrates may in some cases be a remnant of the pre-ubiquitome world, but in other cases could provide optimized regulatory solutions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Dynamics of the intrinsically disordered C-terminal domain of the nipah virus nucleoprotein and interaction with the x domain of the phosphoprotein as unveiled by NMR spectroscopy.

We provide an atomic-resolution description based on NMR spectroscopy, of the intrinsically disordered C-terminal domain of the Nipah virus nucleoprotein (NTAIL ), both in its isolated state and within the nucleocapsid (NC). Within the NC the second half of NTAIL retains conformational behavior similar to that of isolated NTAIL , whereas the first half of NTAIL becomes much more rigid. In spite of the mostly disordered nature of NTAIL , chemical shifts and relaxation measurements show a significant degree of α-helical sampling in the molecular recognition element (MoRE) involved in binding to the X domain (XD) of the phosphoprotein, with this preconfiguration being more pronounced than in the NTAIL domain from the cognate Hendra virus. Outside the MoRE, an additional region exhibiting reduced flexibility was identified within NTAIL and found to be involved in binding to the XD. (1) H- and (13) C-detected titration NMR experiments support a highly dynamic binding of NTAIL at the surface of the XD.

Order and Disorder in the Replicative Complex of Paramyxoviruses.

In this review we summarize available data showing the abundance of structural disorder within the nucleoprotein (N) and phosphoprotein (P) from three paramyxoviruses, namely the measles (MeV), Nipah (NiV) and Hendra (HeV) viruses. We provide a detailed description of the molecular mechanisms that govern the disorder-to-order transition that the intrinsically disordered C-terminal domain (NTAIL) of their N proteins undergoes upon binding to the C-terminal X domain (XD) of the homologous P proteins. We also show that a significant flexibility persists within NTAIL-XD complexes, which therefore provide illustrative examples of "fuzziness". The functional implications of structural disorder for viral transcription and replication are discussed in light of the ability of disordered regions to establish a complex molecular partnership and to confer a considerable reach to the elements of the replicative machinery.

Slippery substrates impair function of a bacterial protease ATPase by unbalancing translocation versus exit.

BACKGROUND: ATP-dependent proteases translocate and unfold their substrates. RESULTS: A human virus sequence with only Gly and Ala residues causes similar dysfunctions of eukaryotic and prokaryotic protease motors: unfolding failure. CONCLUSION: Sequences with amino acids of simple shape and small size impair unfolding of contiguous stable domains. SIGNIFICANCE: Compartmented ATP-dependent proteases of diverse origin share conserved principles of interaction between translocase/effector and substrate/recipient. ATP-dependent proteases engage, translocate, and unfold substrate proteins. A sequence with only Gly and Ala residues (glycine-alanine repeat; GAr) encoded by the Epstein-Barr virus of humans inhibits eukaryotic proteasome activity. It causes the ATPase translocase to slip on its protein track, stalling unfolding and interrupting degradation. The bacterial protease ClpXP is structurally simpler than the proteasome but has related elements: a regulatory ATPase complex (ClpX) and associated proteolytic chamber (ClpP). In this study, GAr sequences were found to impair ClpXP function much as in proteasomes. Stalling depended on interaction between a GAr and a suitably spaced and positioned folded domain resistant to mechanical unfolding. Persistent unfolding failure results in the interruption of degradation and the production of partial degradation products that include the resistant domain. The capacity of various sequences to cause unfolding failure was investigated. Among those tested, a GAr was most effective, implying that viral selection had optimized processivity failure. More generally, amino acids of simple shape and small size promoted unfolding failure. The ClpX ATPase is a homohexamer. Partial degradation products could exit the complex through transient gaps between the ClpX monomers or, alternatively, by backing out. Production of intermediates by diverse topological forms of the hexamer was shown to be similar, excluding lateral escape. In principle, a GAr could interrupt degradation because 1) the translocase thrusts forward less effectively or because 2) the translocase retains substrate less well when resetting between forward strokes. Kinetic analysis showed that the predominant effect was through the second of these mechanisms.

Coiled-coil deformations in crystal structures: the measles virus phosphoprotein multimerization domain as an illustrative example.

The structures of two constructs of the measles virus (MeV) phosphoprotein (P) multimerization domain (PMD) are reported and are compared with a third structure published recently by another group [Communie et al. (2013), J. Virol. 87, 7166-7169]. Although the three structures all have a tetrameric and parallel coiled-coil arrangement, structural comparison unveiled considerable differences in the quaternary structure and unveiled that the three structures suffer from significant structural deformation induced by intermolecular interactions within the crystal. These results show that crystal packing can bias conclusions about function and mechanism based on analysis of a single crystal structure, and they challenge to some extent the assumption according to which coiled-coil structures can be reliably predicted from the amino-acid sequence. Structural comparison also highlighted significant differences in the extent of disorder in the C-terminal region of each monomer. The differential flexibility of the C-terminal region is also supported by size-exclusion chromatography and small-angle X-ray scattering studies, which showed that MeV PMD exists in solution as a dynamic equilibrium between two tetramers of different compaction. Finally, the possible functional implications of the flexibility of the C-terminal region of PMD are discussed.

Functional asymmetries of proteasome translocase pore.

Degradation by proteasomes involves coupled translocation and unfolding of its protein substrates. Six distinct but paralogous proteasome ATPase proteins, Rpt1 to -6, form a heterohexameric ring that acts on substrates. An axially positioned loop (Ar-Φ loop) moves in concert with ATP hydrolysis, engages substrate, and propels it into a proteolytic chamber. The aromatic (Ar) residue of the Ar-Φ loop in all six Rpts of S. cerevisiae is tyrosine; this amino acid is thought to have important functional contacts with substrate. Six yeast strains were constructed and characterized in which Tyr was individually mutated to Ala. The mutant cells were viable and had distinct phenotypes. rpt3, rpt4, and rpt5 Tyr/Ala mutants, which cluster on one side of the ATPase hexamer, were substantially impaired in their capacity to degrade substrates. In contrast, rpt1, rpt2, and rpt6 mutants equaled or exceeded wild type in degradation activity. However, rpt1 and rpt6 mutants had defects that limited cell growth or viability under conditions that stressed the ubiquitin proteasome system. In contrast, the rpt3 mutant grew faster than wild type and to a smaller size, a defect that has previously been associated with misregulation of G1 cyclins. This rpt3 phenotype probably results from altered degradation of cell cycle regulatory proteins. Finally, mutation of five of the Rpt subunits increased proteasome ATPase activity, implying bidirectional coupling between the Ar-Φ loop and the ATP hydrolysis site. The present observations assign specific functions to individual Rpt proteins and provide insights into the diverse roles of the axial loops of individual proteasome ATPases.

Dependence of proteasome processing rate on substrate unfolding.

Protein degradation by eukaryotic proteasomes is a multi-step process involving substrate recognition, ATP-dependent unfolding, translocation into the proteolytic core particle, and finally proteolysis. To date, most investigations of proteasome function have focused on the first and the last steps in this process. Here we examine the relationship between the stability of a folded protein domain and its degradation rate. Test proteins were targeted to the proteasome independently of ubiquitination by directly tethering them to the protease. Degradation kinetics were compared for test protein pairs whose stability was altered by either point mutation or ligand binding, but were otherwise identical. In both intact cells and in reactions using purified proteasomes and substrates, increased substrate stability led to an increase in substrate turnover time. The steady-state time for degradation ranged from ∼5 min (dihydrofolate reductase) to 40 min (I27 domain of titin). ATP turnover was 110/min./proteasome, and was not markedly changed by substrate. Proteasomes engage tightly folded substrates in multiple iterative rounds of ATP hydrolysis, a process that can be rate-limiting for degradation.

Molecular mechanism of NADPH-glyceraldehyde-3-phosphate dehydrogenase regulation through the C-terminus of CP12 in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) consists of four GapA subunits. This A4 GAPDH is not autonomously regulated, as the regulatory cysteine residues present on GapB subunits are missing in GapA subunits. The regulation of A4 GAPDH is provided by another protein, CP12. To determine the molecular mechanisms of regulation of A4 GAPDH, we mutated three residues (R82, R190, and S195) of GAPDH of C. reinhardtii. Kinetic studies of GAPDH mutants showed the importance of residue R82 in the specificity of GAPDH for NADPH, as previously shown for the spinach enzyme. The cofactor NADPH was not stabilized through the 2'-phosphate by the serine 195 residue of the algal GAPDH, unlike the case in spinach. The mutation of R190 also led to a structural change that was not observed in the spinach enzyme. This mutation led to a loss of activity for NADPH and NADH, indicating the crucial role of this residue in maintaining the algal GAPDH structure. Finally, the interaction between GAPDH mutants and wild-type and mutated CP12 was analyzed by immunoblotting experiments, surface plasmon resonance, and kinetic studies. The results obtained with these approaches highlight the involvement of the last residue of CP12, Asp80, in modulating the activity of GAPDH by preventing access of the cofactor NADPH to the active site. These results help us to bridge the gap between our knowledge of structure and our understanding of functional biology in GAPDH regulation.

Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis.

CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion that is involved in the transition of CP12 from a reduced to an oxidized state. In order to describe CP12's copper-binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modelling, were coupled with top-down MS [electrospray-ionization MS and MS/MS (tandem MS)]. Immobilized-copper-ion-affinity-chromatographic experiments allowed the primary characterization of the effects of mutation on copper binding. Top-down MS/MS experiments carried out under non-denaturing conditions on wild-type and mutant CP12-Cu(2+) complexes then allowed fragment ions specifically binding the copper ion to be determined. Comparison of MS/MS datasets defined three regions involved in metal ion binding: residues Asp(16)-Asp(23), Asp(38)-Lys(50) and Asp(70)-Glu(76), with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic acid and aspartic residues, a situation that contrasts with that obtaining for typical protein copper chelators. We propose that copper might play a role in the regulation of the biological activity of CP12.

A new function of GAPDH from Chlamydomonas reinhardtii: a thiol-disulfide exchange reaction with CP12.

CP12 is a flexible protein that is well-known to interact with GAPDH, and this association is crucial to the regulation of enzyme activity. This regulation is likely related to structural transitions of both proteins, but the molecular bases of these changes are not yet understood. To answer this issue, we undertook a study based on the use of paramagnetic probes grafted on cysteine residues and followed by EPR spectroscopy. We present a new application of this approach that enables us to probe the functional role of cysteine residues in protein-protein interactions. Algal CP12 contains four cysteine residues involved in two disulfide bridges in its oxidized state and has some alpha-helical secondary structural elements. In contrast, in its reduced state, CP12 is mainly unstructured and shares some physical properties with intrinsically disordered proteins. Treatment of CP12 with a methane thiosulfonate derivative spin-label (MTSL) led to the labeling of the cysteine residues involved in the C-terminal bridge only as revealed by mass spectrometry. Surprisingly, the partner protein GAPDH induced the cleavage of the disulfide bridge between the cysteine residues of CP12 and the spin-label, resulting in the full release of the label. We showed the existence of a transitory interaction between both proteins and proposed a mechanism based on a thiol-disulfide exchange reaction. The results of this study point out a novel role of the algal GAPDH which is often termed a "moonlighting" protein.

Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex--purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii.

Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6-bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 +/- 0.05 mum and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55+/-5 nm. Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii, we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis-Menten kinetics with fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, with a catalytic constant equal to 35 +/- 1 s(-1) and 4 +/- 0.1 s(-1), respectively. The K(m) value for fructose 1,6-bisphosphate was equal to 0.16 +/- 0.02 mm and 0.046 +/- 0.005 mm for sedoheptulose 1,7-bisphosphate. The catalytic efficiency of aldolase was thus 219 +/- 31 s(-1).mm(-1) with fructose 1,6-bisphosphate and 87 +/- 9 s(-1).mm(-1) with sedoheptulose 1,7-bisphosphate. In the presence of the complex, this parameter for fructose 1,6-bisphosphate increased to 310 +/- 23 s(-1).mm(-1), whereas no change was observed with sedoheptulose 1,7-bisphosphate. The condensation reaction of aldolase to form fructose 1,6-bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.

Hypermethylation of 28S ribosomal RNA in ß-thalassemia trait carriers.

Ribosome biogenesis is the process of synthesis of the cellular ribosomes which mediate protein translation. Integral with the ribosomes are four cytoplasmic ribosomal RNAs (rRNAs) which show extensive post-transcriptional modifications including 2'-O-methylation and pseudouridylation. Several hereditary hematologic diseases including Diamond-Blackfan anemia have been shown to be associated with defects in ribosome biogenesis. Thalassemia is the most important hematologic inherited genetic disease worldwide, and this study examined the post-transcriptional ribose methylation status of three specific active sites of the 28S rRNA molecule at positions 1858, 4197 and 4506 of ß-thalassemia trait carriers and normal controls. Samples from whole blood and cultured erythroid cells were examined. Results showed that site 4506 was hypermethylated in ß-thalassemia trait carriers in both cohorts. Expression of fibrillarin, the ribosomal RNA methyltransferase as well as snoRNAs were additionally quantified by RT-qPCR and evidence of dysregulation was seen. Hemoglobin E trait carriers also showed evidence of dysregulation. These results provide the first evidence that ribosome biogenesis is dysregulated in ß-thalassemia trait carriers.

Demonstration of a folding after binding mechanism in the recognition between the measles virus NTAIL and X domains.

In the past decade, a wealth of experimental data has demonstrated that a large fraction of proteins, while functional, are intrinsically disordered at physiological conditions. Many intrinsically disordered proteins (IDPs) undergo a disorder-to-order transition upon binding to their biological targets, a phenomenon known as induced folding. Induced folding may occur through two extreme mechanisms, namely conformational selection and folding after binding. Although the pre-existence of ordered structures in IDPs is a prerequisite for conformational selection, it does not necessarily commit to this latter mechanism, and kinetic studies are needed to discriminate between the two possible scenarios. So far, relatively few studies have addressed this issue from an experimental perspective. Here, we analyze the interaction kinetics between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein. Data reveal that NTAIL recognizes XD by first forming a weak encounter complex in a disordered conformation, which is subsequently locked-in by a folding step; i.e., binding precedes folding. The implications of our kinetic results, in the context of previously reported equilibrium data, are discussed. These results contribute to enhancing our understanding of the molecular mechanisms by which IDPs recognize their partners and represent a paradigmatic example of the need of kinetic methods to discriminate between reaction mechanisms.

Insights into the coiled-coil organization of the Hendra virus phosphoprotein from combined biochemical and SAXS studies.

Nipah and Hendra viruses are recently emerged paramyxoviruses belonging to the Henipavirus genus. The Henipavirus phosphoprotein (P) consists of a large intrinsically disordered domain and a C-terminal domain (PCT) containing alternating disordered and ordered regions. Among these latter is the P multimerization domain (PMD). Using biochemical, analytical ultracentrifugation and small-angle X-ray scattering (SAXS) studies, we show that Hendra virus (HeV) PMD forms an elongated coiled-coil homotrimer in solution, in agreement with our previous findings on Nipah virus (NiV) PMD. However, the orientation of the N-terminal region differs from that observed in solution for NiV PMD, consistent with the ability of this region to adopt different conformations. SAXS studies provided evidence for a trimeric organization also in the case of PCT, thus extending and strengthening our findings on PMD. The present results are discussed in light of conflicting reports in the literature pointing to a tetrameric organization of paramyxoviral P proteins.

Biochemical and structural studies of the oligomerization domain of the Nipah virus phosphoprotein: evidence for an elongated coiled-coil homotrimer.

Nipah virus (NiV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The NiV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the polymerase for transcription and replication. The polymerase is recruited onto the nucleocapsid via its cofactor, the phosphoprotein (P). The NiV P protein has a modular organization, with alternating disordered and ordered domains. Among these latter, is the P multimerization domain (PMD) that was predicted to adopt a coiled-coil conformation. Using both biochemical and biophysical approaches, we show that NiV PMD forms a highly stable and elongated coiled-coil trimer, a finding in striking contrast with respect to the PMDs of Paramyxoviridae members investigated so far that were all found to tetramerize. The present results therefore represent the first report of a paramyxoviral P protein forming trimers.

Dynamics of the intrinsically disordered protein CP12 in its association with GAPDH in the green alga Chlamydomonas reinhardtii: a fuzzy complex.

CP12 is a widespread regulatory protein of oxygenic photosynthetic organisms that contributes to the regulation of the Calvin cycle by forming a supra-molecular complex with at least two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). CP12 shares some similarities with intrinsically disordered proteins (IDPs) depending on its redox state. In this study, site-directed spin labeling (SDSL) combined with EPR spectroscopy was used to probe the dynamic behavior of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH, the first step towards ternary complex formation. The two N-terminal cysteine residues were labeled using the classical approach while the tyrosine located at the C-terminal end of CP12 was modified following an original procedure. The results show that the label grafted at the C-terminal extremity is in the vicinity of the interaction site whereas the N-terminal region remains fully disordered upon binding to GAPDH. In conclusion, GAPDH-CP12 is a fuzzy complex, in which the N-terminal region of CP12 keeps a conformational freedom in the bound form. This fuzziness could be one of the keys to facilitate binding of PRK to CP12-GAPDH and to form the ternary supra-molecular complex.