Structural and biophysical studies of RNA-Chaperone Hfq from E. coli

Diese Dissertation wurde an der Universität Wien in den Max F. Perutz Laboratories von Mag. Mads Beich-Frandsen von 2005 bis 2011 durchgeführt und betreut von Prof. Kristina Djinović-Carugo vom Department für Strukturbiologie und Computational Biology in Kollaboration mit Prof. Udo Bläsi vom Department für Mikrobiologie, Immunbiologie und Genetik. Der Titel verweist auf die strukturbiologischen Untersuchungen am RNA-bindenden Protein Hfq aus Escherichia coli. Durch die Erkenntnis, dass nur ein Bruchteil des gesamten Genoms für Protein kodiert, verlagerte sich der Forschungsschwerpunkt in der Biologie hin zu RNA-basierter Regulation. Mit der wachsenden Anzahl vollständiger Transkriptomprofile kristallisiert sich das Sm-like Protein Hfq als zentrale Schaltstelle zur Genregulation durch small regulatory RNAs (sRNAs) in Bakterien heraus. In E. coli und anderen gram-negativen Pathogenen ist der konservierte Sm-like Kern von Hfq um eine carboxyterminale Domäne erweitert, deren Länge 30% der Sequenz des gesamten Proteins beträgt. Seine kurze aminoterminale Region ist hingegen in höherem Maße konserviert. Sowohl N- als auch C-Terminus von Hfq, beide gekennzeichnet durch intrinsisches Fehlen einer geordneten Struktur, tragen nachweislich zur Funktionalität des Proteins bei. Der Schlüsselmechanismus der Hfq-vermittelten Regulation besteht darin, transkodierte sRNAs mit ihrer Ziel-mRNA zu hybridisieren. Hfq agiert somit als RNA-Chaperon, welches die Sekundärstruktur von RNA modifiziert. Die Aufgabenstellung des Projekts bestand darin, die Funktion von Hfq aus einer strukturbiologischen Perspektive zu beleuchten. Im Rahmen dieser Forschungsarbeit wurden Röntgenkristallographie, Kleinwinkelstreuung, NMR Spektroskopie, Zirkulardichroismus mit Synchrotronstrahlung, kombiniert mit und integriert in bioinformatische und funktionelle Studien, angewendet. Aus dieser Arbeit gingen zwei Publikationen hervor, die strukturelle Aspekte von Hfq in E. coli beschreiben. Die Analyse jener Ergebnisse geschah im Kontext biophysikalischer und funktioneller Resultate, welche den intrinisch unstrukturierten Termini von E. coli Hfq Funktionalität zuweisen. Es konnte festgestellt werden, dass die Termini, ausgelöst durch die Interaktion mit RNA, Strukturen ausbilden. Die Interpretation dieser Resultate folgt dem „Entropietransfermodell“, welches vorschlägt, dass intrinisch unstrukturierte Sequenzen durch isothermische Enthalpie/Entropie-Kompensation die Entfaltung von Zielstrukturen begünstigen können. Das Zusammenspiel von strukturierter und ungeordeter Sequenz in E.coli Hfq ermöglicht es diesem Protein, mit einer Vielzahl an RNAs zu interagieren und diese zu regulieren. Die zentrale Funktion von Hfq ist hierbei, die RNA in einem ungefalteten Zustand zu erhalten. Sie kann in folgendem Dogma zusammengefasst werden: Bindung fördert Entfaltung – Entfaltung fördert Hybridisierung - Hybridisierung fördert Loslösung von Hfq!This dissertation was conducted at the University of Vienna, Max F. Perutz Laboratories in the years 2005-2011. Here is reported on research performed by Mag. Mads Beich-Frandsen, supervised by Prof. Kristina Djinovic-Carugo at the Department of Structural and Computational Biology, in collaboration with Prof. Udo Bläsi at the Department of Microbiology, Immunobiology and Genetics. The title refers to the biostructural investigations conducted for the RNA-binding protein Hfq from Escherichia coli. Upon the understanding that only a fraction of a genome encodes protein, focus has been shifted to RNA-based regulation in biology. With the increasing number of transcriptome profiles being completed, the Sm-like protein Hfq emerges as the central switchboard of gene regulation, as mediated by small regulatory RNAs (sRNAs) in Bacteria. In E. coli and other gram-negative pathogens, the conserved Sm-like core of Hfq is extended 30% in sequence length, by a C-terminal domain. The short N-terminal region of Hfq is conserved to higher degree. Both the N- and C-terminus of Hfq have been demonstrated of functional importance for the protein, and are characterized as intrinsically disordered. The key mechanism of Hfq-mediated regulation is by annealing trans-encoded sRNAs to target mRNA. Here Hfq acts as an RNA-chaperone, with ability to alter the secondary structure of RNA. The scope of the project was to elucidate the function of E. coli Hfq from the perspective of structural biology. The research presented here employs X-ray crystallography, Small Angle Scattering, Nuclear Magnetic Resonance, Synchrotron-Radiation Circular-Dichroism, in an integrated approach with bioinformatics and functional studies. The work resulted in two publications, reporting on structural aspects of E. coli Hfq. These results were analyzed in context of acquired biophysical and functional results, which annotates function to the intrinsically disordered N- and C-terminus of E. coli Hfq. Interaction with RNA was found to induce structure upon the termini of Hfq. This was interpreted in line of the ‘entropy-transfer’ model, which proposes intrinsically disordered sequence to have a function in unfolding targets by isothermal entropy/enthalpy compensation. The interplay between the structured and disordered sequence in E. coli Hfq provides the protein with the ability to interact with and exert regulation on a wide variety of RNAs. Hfq functions to keep the RNA unfolded, following the dogma: Binding promotes unfolding – unfolding promotes annealing – annealing promotes release of Hfq

Study of E. coli Hfq's RNA annealing acceleration and duplex destabilization activities using substrates with different GC-contents

Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq’s activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq’s strand displacement as well as its annealing activity are strongly dependent on the substrate’s GC-content. However, this is due to Hfq’s preferred binding of AU-rich sequences and not to the substrate’s thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq’s strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq’s thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other

Translational activation of rpoS mRNA by the non-coding RNA DsrA and Hfq does not require ribosome binding

At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, σS, involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind

Structure of the N-terminal domain of the protein Expansion: An 'Expansion' to the Smad MH2 fold

Supporting information at doi:10.1107/S1399004715001443/kw5112sup1.pdfGene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.This work was supported by the Spanish National Research Program (MINECO, SAF2011-25119). MBF was supported by a Marie Curie Action (COFUND) within the European Union Seventh Framework Programme. MJM is an ICREA Programme InvestigatorPeer Reviewe

Structural and biochemical studies on ATP binding and hydrolysis by the Escherichia coli RNA chaperone Hfq.

In Escherichia coli the RNA chaperone Hfq is involved in riboregulation by assisting base-pairing between small regulatory RNAs (sRNAs) and mRNA targets. Several structural and biochemical studies revealed RNA binding sites on either surface of the donut shaped Hfq-hexamer. Whereas sRNAs are believed to contact preferentially the YKH motifs present on the proximal site, poly(A)(15) and ADP were shown to bind to tripartite binding motifs (ARE) circularly positioned on the distal site. Hfq has been reported to bind and to hydrolyze ATP. Here, we present the crystal structure of a C-terminally truncated variant of E. coli Hfq (Hfq(65)) in complex with ATP, showing that it binds to the distal R-sites. In addition, we revisited the reported ATPase activity of full length Hfq purified to homogeneity. At variance with previous reports, no ATPase activity was observed for Hfq. In addition, FRET assays neither indicated an impact of ATP on annealing of two model oligoribonucleotides nor did the presence of ATP induce strand displacement. Moreover, ATP did not lead to destabilization of binary and ternary Hfq-RNA complexes, unless a vast stoichiometric excess of ATP was used. Taken together, these studies strongly suggest that ATP is dispensable for and does not interfere with Hfq-mediated RNA transactions

The purine binding site between adjacent subunits on the distal face of <i>E. coli</i> Hfq.

<p>Side-chains of binding site residues are shown by a stick. A single ATP-molecule is depicted with the triple-phosphate protruding in the favored conformation (transparent orange stick). Inset: The solvent accessible area of Hfq hexamer, colored according to its electrostatic potential (red and blue correspond to negatively and positively charged residues, respectively), is shown from the distal face with four ATP molecules bound.</p

Structures of the ligand-binding core of iGluR2 in complex with the agonists (R)- and (S)-2-amino-3-(4-hydroxy-1,2,5-thiadiazol-3-yl)propionic acid explain their unusual equipotency

AMPA type ionotropic glutamate receptors generally display high stereoselectivity in agonist binding. However, the stereoisomers of 2 amino 3 4 hydroxy 1,2,5 thiadiazol 3 yl propionic acid TDPA have similar enantiopharmacology. To understand this observation, we have determined the X ray structures of R TDPA and S TDPA in complex with the ligand binding core of iGluR2 and investigated the binding pharmacology at AMPA and kainate receptors. Both enantiomers induce full domain closure in iGluR2 but adopt different conformations when binding to the receptor, which may explain the similar enantiopharmacolog

Interactions of adenines in Hfq-nucleotide complexes.

<p>(<b>A</b>) Hydrogen bond <b>i</b>nteraction between ribose 2′-OH with the Gly-29 carbonyl atom, for which a specific main chain conformation is required. ATP is shown as balls and sticks with the following color code for atoms: C – yellow; N - blue; O - red; P – orange. Hfq₆₅ residues involved in the interaction are presented by a stick. (<b>B</b>) Superposition of Hfq_Bs-AGAGAG (<i>B. subtilis</i> Hfq), Hfq-ADP (<i>E. coli</i> Hfq), Hfq-polyA (<i>E. coli</i> Hfq), Hfq_Pa-ADPNP (<i>P. aeruginosa</i> Hfq) and Hfq₆₅-ATP complexes to highlight the spread of purine ring orientations around the normal to the ring. Blue and red circles enclose two main clusters: in the first the exocyclic N6 atom hydrogen bonds to the Gln-52′ side-chain, in the second to the Thr-61 side-chain. N6 atoms are depicted as spheres. The colour code for atoms in the first cluster is: C - cyan; N - blue; O - red; P – orange; in the second C - yellow, and in the outlier ligand of the Hfq_Pa-ADPNP complex C - green.</p