The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases

Emg1 was previously shown to be required for maturation of the 18S rRNA and biogenesis of the 40S ribosomal subunit. Here we report the determination of the crystal structure of Emg1 at 2 Å resolution in complex with the methyl donor, S-adenosyl-methionine (SAM). This structure identifies Emg1 as a novel member of the alpha/beta knot fold methyltransferase (SPOUT) superfamily. In addition to the conserved SPOUT core, Emg1 has two unique domains that form an extended surface, which we predict to be involved in binding of RNA substrates. A point mutation within a basic patch on this surface almost completely abolished RNA binding in vitro. Three point mutations designed to disrupt the interaction of Emg1 with SAM each caused>100-fold reduction in SAM binding in vitro. Expression of only Emg1 with these mutations could support growth and apparently normal ribosome biogenesis in strains genetically depleted of Emg1. We conclude that the catalytic activity of Emg1 is not essential and that the presence of the protein is both necessary and sufficient for ribosome biogenesis

Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation

Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C- domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins

Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses

The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues) encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes

The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity

The 5-methyluridine is invariably found at position 54 in the TΨC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase (PabTrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of PabTrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 Å resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of PabTrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by PabTrmU54. These results were used to model yeast tRNAAsp in the PabTrmU54 structure to get further insights into the different RNA specificities of RumA and PabTrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to PabTrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TΨC loop conformational change has to occur for the flipping of the target uridine into the PabTrmU54 active site during catalysis

Crystal Structure of the PP2A Phosphatase Activator: Implications for Its PP2A-Specific PPIase Activity

PTPA, an essential and specific activator of protein phosphatase 2A (PP2A), functions as a peptidyl prolyl isomerase (PPIase). We present here the crystal structures of human PTPA and of the two yeast orthologs (Ypa1 and Ypa2), revealing an all α-helical protein fold that is radically different from other PPIases. The protein is organized into two domains separated by a groove lined by highly conserved residues. To understand the molecular mechanism of PTPA activity, Ypa1 was cocrystallized with a proline-containing PPIase peptide substrate. In the complex, the peptide binds at the interface of a peptide-induced dimer interface. Conserved residues of the interdomain groove contribute to the peptide binding site and dimer interface. Structure-guided mutational studies showed that in vivo PTPA activity is influenced by mutations on the surface of the peptide binding pocket, the same mutations that also influenced the in vitro activation of PP2Ai and PPIase activity

Evf, a virulence factor produced by the Drosophila pathogen Erwinia carotovora, is an S-palmitoylated protein with a new fold that binds to lipid vesicles

Erwinia carotovora are phytopathogenic Gram-negative bacteria of agronomic interest as these bacteria are responsible for fruit soft rot and use insects as dissemination vectors. The Erwinia carotovora carotovora strain 15 (Ecc15) is capable of persisting in the Drosophila gut by the sole action of one protein, Erwinia virulence factor (Evf). However, the precise function of Evf is elusive, and its sequence does not provide any indication as to its biochemical function. We have solved the 2.0-angstroms crystal structure of Evf and found a protein with a complex topology and a novel fold. The structure of Evf confirms that Evf is unlike any virulence factors known to date. Most remarkably, we identified palmitoic acid covalently bound to the totally conserved Cys209, which provides important clues as to the function of Evf. Mutation of the palmitoic binding cysteine leads to a loss of virulence, proving that palmitoylation is at the heart of Evf infectivity and may be a membrane anchoring signal. Fluorescence studies of the sole tryptophan residue (Trp94) demonstrated that Evf was indeed able to bind to model membranes containing negatively charged phospholipids and to promote their aggregation

Etude structurale et fonctionnelle du sous-complexe Fap7-Rps14 impliqué dans la biogenèse du ribosome

Plus de 200 facteurs pré-ribosomiques sont impliqués dans la maturation des ribosomes. La majorité de ces facteurs sont essentiels à la survie cellulaire, mais la fonction précise de la plupart d entre eux demeure inconnue. Une des dernières étapes de maturation de la petite sous-unité du ribosome est le clivage du pré-ARNr 20S en ARNr 18S mature. Ce clivage est réalisé par l'endonucléase Nob1 et nécessite également la présence de la NTPase Fap7 ainsi que d une pléthore d autres facteurs pré-ribosomiques. La fonction de Fap7 est particulièrement intrigante, car l'homologue humain hCINAP possède une activité adénylate kinase, activité enzymatique qui n est généralement pas liée à la biogenèse des particules ribonucléoprotéiques. En outre, la fonction de Fap7 est intimement liée à son interaction avec la protéine ribosomique Rps14. La partie C-terminale de Rps14 est essentielle pour le clivage au niveau du site D et est située à proximité de l extrémité 3 de l ARNr 18S dans le ribosome mature. La suppression de cette protéine provoque le syndrome 5q qui est phénotypiquement proche de l anémie de Diamond-Blackfan. Ces deux protéines interviennent également au niveau d une voie de régulation de p53 qui est dérégulée dans de nombreux cancers. La combinaison d études structurales par cristallographie aux rayons X, d études enzymatiques sur des protéines recombinantes ainsi que des tests de maturation in vitro réalisés sur des pré-ribosomes purifiés, nous a permis de mieux appréhender la fonction de Fap7 au sein de la sous-unité pré-40S du ribosome. Nous avons également montré que l'interaction Fap7-Rps14 est impliquée dans un changement conformationnel majeur au cœur des pré-ribosomes et que cette réorganisation est nécessaire afin d'exposer le site D pour le clivage par l endonucléase Nob1.Over 200 pre-ribosomal factors involved in the maturation of ribosomes. Most of these factors are essential to cell survival, but the precise function of most of these factors remains elusive. One of the last steps of maturation of the small subunit of the ribosome is the cleavage of 20S pre-rRNA in 18S rRNA in the cytoplasm. This cleavage is carried out by the endonuclease Nob1 and also requires the presence of other factors such as the methyltransferase Dim1, and a plethora of NTPases including the Rio protein kinases, Prp43 and its cofactor Pfa1, the Ltv1 GTPase and the Fap7 NTPase. The function of Fap7 is especially intriguing since the human homologue bears Adenylate activity, an enzymatic activity not usually linked to ribonucleoprotein biogenesis. In addition, the function of Fap7 is intimately linked its interaction with the Rps14 ribosomal protein. The Rps14 C-terminal is essential of D site cleavage and is located in proximity to the 18S C-terminus in the mature ribosome. The deletion of this protein causes the 5q syndrome that is phenotypically close to Diamond Blackfan anemia. The link between the enzymatic activity of Fap7 and its role in ribosome biogenesis remains enigmatic. Using a combination of structural studies by X-ray crystallography, small angle X-ray scattering (SAXS) in solution, enzymatic studies on purified proteins, and in vitro D site cleavage reaction assays on purified pre-ribosomes, we were able to uncover the function of Fap7 within pre-40S ribosomes. We show that the Fap7/Rps14 interaction is involved in a major conformational change at the heart of the pre-ribosomes and that this structural rearrangement is necessary to expose the D-site for cleavage by the endonuclease Nob1.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. électronique (751069903) / SudocSudocFranceF

La synthèse des ribosomes, au cœur du contrôle de la prolifération cellulaire

Les ribosomes sont au cœur de l’expression génique. Leur synthèse suit un processus complexe et coûteux en énergie qui fait l’objet de nombreux contrôles afin d’assurer à la cellule un taux de production de ribosomes optimal. Dans cette revue, nous détaillons les récentes découvertes associant des perturbations de l’assemblage des ribosomes au contrôle du cycle cellulaire, notamment par l’intermédiaire du suppresseur de tumeur p53. La particule 5S, un sous-complexe ribosomique, est au cœur de cette régulation qui s’intègre à d’autres signaux contrôlant la progression dans le cycle cellulaire tels que p14ARF, SRSF1 ou PRAS40. Ces données font de l’assemblage des ribosomes une nouvelle cible thérapeutique pour le traitement de certains cancers