Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer

The genome of influenza A virus is organized into eight ribonucleoprotein complexes (RNPs), each containing one RNA polymerase complex. This RNA polymerase has also been found non-associated to RNPs and is possibly involved in distinct functions in the infection cycle. We have expressed the virus RNA polymerase complex by co-tranfection of the PB1, PB2 and PA genes in mammalian cells and the heterotrimer was purified by the TAP tag procedure. Its 3D structure was determined by electron microscopy and single-particle image processing. The model obtained resembles the structure previously reported for the polymerase complex associated to viral RNPs but appears to be in a more open conformation. Detailed model comparison indicated that specific areas of the complex show important conformational changes as compared to the structure for the RNP-associated polymerase, particularly in regions known to interact with the adjacent NP monomers in the RNP. Also, the PB2 subunit seems to undergo a substantial displacement as a result of the association of the polymerase to RNPs. The structural model presented suggests that a core conformation of the polymerase in solution exists but the interaction with other partners, such as proteins or RNA, will trigger distinct conformational changes to activate new functional properties

The C-terminal SH3 domain contributes to the intramolecular inhibition of Vav family proteins

Vav proteins are phosphorylation-dependent guanine nucleotide exchange factors (GEFs) that catalyze the activation of members of the Rho family of guanosine triphosphatases (GTPases). The current regulatory model holds that the nonphosphorylated, catalytically inactive state of these GEFs is maintained by intramolecular interactions among the amino-terminal domains and the central catalytic core, which block the binding of Vav proteins to GTPases. We showed that this autoinhibition is mechanistically more complex, also involving the bivalent association of the carboxyl-terminal Src homology 3 (SH3) region of Vav with its catalytic and pleckstrin homology (PH) domains. Such interactions occurred through proline-rich region-independent mechanisms. Full release from this double-locked state required synergistic weakening effects from multiple phosphorylated tyrosine residues, thus providing an optimized system to generate gradients of Vav GEF activity depending on upstream signaling inputs. This mechanism is shared by mammalian and Drosophila melanogaster Vav proteins, suggesting that it may be a common regulatory feature for this protein family.Work in the laboratory of X.R.B. has been funded by grants from the Spanish Ministry of Economy and Competitiveness (SAF2009-07172, SAF2012-3171, RD06/0020/0001, and RD12/0036/0002), the Castilla-León Autonomous Government (CSI039A12-1), and the Asociación Española Contra el Cáncer (AECC). O.L. has been supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2011-22988 and RD06/0020/1001). The salary of M.B. has been partially supported by a JAE-Predoc contract (CSIC), the AECC, and grant RD06/0020/0001. S.F. is supported by a graduate student FPI contract from the Spanish Ministry of Economy and Competitiveness (BES-2010-031386). Spanish funding is cosponsored by the European Regional Development Fund.Peer Reviewe

The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.The project was supported by grant BFU2013-48374-P of the Spanish MINECO and by the Ramón Areces Foundation. O.G. held a research contract under the Ramón y Cajal program of the Spanish MINECO (RYC-2011-07967). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from the Spanish MINECO.Peer reviewe

Estructura tridimensional de complejos heterotriméricos recombinantes de la polimerasa del virus de la gripe

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 16-5

Crystal structure of Hcp from Acinetobacter baumannii: a component of the type VI secretion system

16 p.-6 fig.-1 tab.The type VI secretion system (T6SS) is a bacterial macromolecular machine widely distributed in Gram-negative bacteria, which transports effector proteins into eukaryotic host cells or other bacteria. Membrane complexes and a central tubular structure, which resembles the tail of contractile bacteriophages, compose the T6SS. One of the proteins forming this tube is the hemolysin co-regulated protein (Hcp), which acts as virulence factor, as transporter of effectors and as a chaperone. In this study, we present the structure of Hcp from Acinetobacter baumannii, together with functional and oligomerization studies. The structure of this protein exhibits a tight β barrel formed by two β sheets and flanked at one side by a short α-helix. Six Hcp molecules associate to form a donut-shaped hexamer, as observed in both the crystal structure and solution. These results emphasize the importance of this oligomerization state in this family of proteins, despite the low similarity of sequence among them. The structure presented in this study is the first one for a protein forming part of a functional T6SS from A. baumannii. These results will help us to understand the mechanism and function of this secretion system in this opportunistic nosocomial pathogen.Peer reviewe

Analysis of the interaction of influenza virus polymerase complex with human cell factors

12 pages, 4 figures.-- PMID: 18491320 [PubMed].-- Supplementary information (Suppl. figure S1, 2 pages) available at: http://www.wiley-vch.de/contents/jc_2120/2008/pro200700508_s.pdfThe influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. Here we present the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. The virus polymerase was expressed by cotransfection of the polymerase subunits cDNAs, one of which fused to the tandem-affinity purification (TAP) tag. The intracellular complexes were purified by the TAP approach, which involves IgG-Sepharose and calmodulin-agarose chromatography, under very mild conditions. The purified complexes contained the heterotrimeric polymerase and a series of associated proteins that were not apparent in purifications of untagged polymerase used as a control. Several influenza polymerase-associated proteins were identified by MALDI-MS and their presence in purified polymerase-containing complexes were verified by Western blot. Their relevance for influenza infection was established by colocalisation with virus ribonucleoproteins in human infected cells. Most of the associated human factors were nuclear proteins involved in cellular RNA synthesis, modification and nucleo-cytoplasmic export, but some were cytosolic proteins involved in translation and transport. The interactions recognised in this proteomic approach suggest that the influenza polymerase might be involved in steps of the infection cycle other than RNA replication and transcription.N. J. was a fellow from Ministerio de Educación y Ciencia. E. T. was a fellow from Instituto de Salud Carlos III. P. G. was a fellow from Gobierno Vasco. This work was supported by the Spanish Ministry of Education and Science (Ministerio de Educación y Ciencia) (grant BFU2004-491), the VIRHOST Program financed by Comunidad de Madrid, European Vigilance Network for the Management of Antiviral Drug Resistance (VIRGIL) and the FLUPOL strep project (SP5B-CT-2007-044263).Peer reviewe

Structure of Yin Yang 1 oligomers that cooperate with RuvBL1-RuvBL2 ATPases

17 p.-9 fig.-2 tab.Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood. Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1. Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination.This work was supported by the Spanish Government (SAF2011-22988 (to O. L.), BES-2009-014133 (to A. L.-P.), BFU2012-39879-C02-02 (to S. A.)) and by Cancer Research UK (CEA-C7905 (to J. A. D.)).Peer reviewe

Structural studies on RNA polymerase I transcription

Resumen del póster presentado al XXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015.Biosynthesis of the eukaryotic ribosome starts with ribosomal RNA production by RNA polymerase I (Pol I), a process that is critical to regulate cell growth and proliferation. We were able to obtain the crystal structure of yeast Pol I, a 14-subunit complex with a total mass of 590,000 Da, at 3.0 Å resolution. The structure represents the latent state of the enzyme, characterized by three major features. First, it forms dimers that involve the C-terminal tail of the stalk subunit A43. Second, the two enzyme halves pivot along the DNA-binding cleft to produce an open cleft and an unfolded bridge helix. Third, an extended loop in subunit A190 mimics the DNA backbone along the cleft, hampering nucleic acid binding. The Pol I crystal structure also reveals intrinsic modules that only bind transiently in other RNA polymerases, such as a TFIIS-like zinc ribbon in subunit A12.2 and a TFIIF-like dimerization module in the A49/A34.5 heterodimer. The Pol I crystal structure will be presented in the light of recent biochemical results.Peer Reviewe

Structure of RepA-WH1 amyloid filaments

Most available structures of amyloids correspond to peptide fragments that self-assemble in extended cross b sheets. However, structures in which a whole protein domain acts as building block of an amyloid fiber are scarce, in spite of their relevance to understand amyloidogenesis. Here, we use electron microscopy (EM) and atomic force microscopy (AFM) to analyze the structure of amyloid filaments assembled by RepA-WH1, a winged-helix domain from a DNA replication initiator in bacterial plasmids. RepA-WH1 functions as a cytotoxic bacterial prionoid that recapitulates features of mammalian amyloid proteinopathies. RepA are dimers that monomerize at the origin to initiate replication, and we find that RepA-WH1 reproduces this transition to form amyloids. RepA-WH1 assembles double helical filaments by lateral association of a singlestranded precursor built by monomers. Double filaments then associate in mature fibers. The intracellular and cytotoxic RepA-WH1 aggregates might reproduce the hierarchical assembly of human amyloidogenic proteins.This work was supported by grants of the Spanish Government SAF2011- 22988 and the Autonomous Region of Madrid S2010/BMD-2316 to O.L.; BIO2012-30852 and CSD2009-00088 to R.G.; BFU2011-25090 to J.M.-B.; and FIS2011-24638 and ERC Starting grant ref 206117 to F.M.-H.Peer reviewe