The structures of mutant forms of Hfq from<i>Pseudomonas aeruginosa</i>reveal the importance of the conserved His57 for the protein hexamer organization

Moskaleva, O.; Melnik, Bogdan S.; Габдулхаков, А.Г.; Garber, Maria; Nikonov, S.V.; Stolboushkina, Elena; Nikulin, Alexei

doi:10.1107/s1744309110017331

Cited by 25 publications

(20 citation statements)

References 31 publications

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“…1d). The importance of this region of the subunit interface was previously demonstrated by amino acid substitutions of H57 in Pseudomonas aeruginosa Hfq, which were also highly destabilizing 22 . Our results raise the possibility that the RNA recognition is impaired in proximal face mutants in part because the hexamer itself is unstable.…”

Section: Resultsmentioning

confidence: 81%

Hexamer to Monomer Equilibrium of E. coli Hfq in Solution and Its Impact on RNA Annealing

Panja

Woodson

2012

Journal of Molecular Biology

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The bacterial Sm-like protein Hfq forms a ring shaped homo-hexamer that is necessary for Hfq to bind nucleic acids and to act in sRNA regulation. Using semi-native gels and fluorescence anisotropy, we show that Hfq undergoes a cooperative conformational change from monomer to hexamer around 1 μM protein, which is comparable to the in vivo concentration of Hfq and above the dissociation constant of the Hfq hexamer from many RNA substrates. Above 2 μM protein, Hfq hexamers associate in high molecular weight complexes. Mutations that impair RNA binding to the proximal face strongly destabilize the hexamer, while the mutation R16A near the outer rim prevents hexamer association. Stopped-flow FRET experiments showed that Hfq subunits interact within a few seconds, suggesting that Hfq monomers, hexamers and multi-hexamer complexes are in dynamic equilibrium. Finally, we show that Hfq is most active in RNA annealing when the hexamer is present. These results suggest that RNA binding is coupled to hexamer assembly, and that the biochemical activity of Hfq reflects the equilibrium between different quaternary structures.

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Section: Resultsmentioning

confidence: 81%

Hexamer to Monomer Equilibrium of E. coli Hfq in Solution and Its Impact on RNA Annealing

Panja

Woodson

2012

Journal of Molecular Biology

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“…This broad specificity would prevent cells from circumventing drug activity by expressing an sRNA with redundant function and would allow a drug to be used on species that employ Hfq for virulence factor production without knowledge of the particular sRNA that is important. The predicted interaction with H57 Hfq would also limit the acquisition of resistance through mutations in Hfq, because H57 Hfq is required for Hfq oligomerization as well as sRNA binding (45), although mutations to other residues could potentially decrease RI20 binding and lead to resistance. A small molecule that binds to the same site as RI20 would be a candidate anti-infective.…”

Section: Figmentioning

confidence: 99%

Cell-Based Assay To Identify Inhibitors of the Hfq-sRNA Regulatory Pathway

El-Mowafi

Alumasa

Ades

et al. 2014

Antimicrob Agents Chemother

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Noncoding small RNAs (sRNAs) act in conjunction with the RNA chaperone Hfq to regulate gene expression in bacteria. Because Hfq is required for virulence in several bacterial pathogens, the Hfq-sRNA system is an attractive target for antibiotic development. A reporter strain in which the expression of yellow fluorescent protein (YFP) is controlled by Hfq-sRNA was engineered to identify inhibitors of this system. A reporter that is targeted by Hfq in conjunction with the RybB sRNA was used in a genetic screen to identify inhibitors from a library of cyclic peptides produced in Escherichia coli using split-intein circular ligation of peptides and proteins (SICLOPPS), an intein-based technology. One cyclic peptide identified in this screen, RI20, inhibited Hfqmediated repression of gene expression in conjunction with both RybB and an unrelated sRNA, MicF. Gel mobility shift assays showed that RI20 inhibited binding of Hfq to RybB and MicF with similar K i values. These data suggest that RI20 inhibits Hfq activity by blocking interactions with sRNAs and provide a paradigm for inhibiting virulence genes in Gram-negative pathogens.

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“…This highly conserved residue is crucial for the stability of the Hfq ring (24) and donates a hydrogen bond from its ϵN nitrogen to the I59* carbonyl oxygen of the neighboring monomer (Fig. 2E).…”

Section: High-resolution Crystal Structure Of St Hfq Bound To a Hexaumentioning

confidence: 99%

Structural basis for RNA 3′-end recognition by Hfq

Sauer

Weichenrieder

2011

Proc. Natl. Acad. Sci. U.S.A.

190

251

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The homohexameric (L)Sm protein Hfq is a central mediator of small RNA-based gene regulation in bacteria. Hfq recognizes small regulatory RNAs (sRNAs) specifically, despite their structural diversity. This specificity could not be explained by previously described RNA-binding modes of Hfq. Here we present a distinct and preferred mode of Hfq-RNA interaction that involves the direct recognition of a uridine-rich RNA 3′ end. This feature is common in bacterial RNA transcripts as a consequence of Rho-independent transcription termination and hence likely contributes significantly to the general recognition of sRNAs by Hfq. Isothermal titration calorimetry shows nanomolar affinity between Salmonella typhimurium Hfq and a hexauridine substrate. We determined a crystal structure of the complex that reveals a constricted RNA backbone conformation in the proximal RNA-binding site of Hfq, allowing for a direct protein contact of the 3′ hydroxyl group. A free 3′ hydroxyl group is crucial for the high-affinity interaction with Hfq also in the context of a full-length sRNA substrate, RybB. The capacity of Hfq to occupy and sequester the RNA 3′ end has important implications for the mechanisms by which Hfq is thought to affect sRNA stability, turnover, and regulation.RNA chaperone | regulation of translation | RNA degradation | prokaryotes H fq is an abundant and widely conserved RNA-binding protein in bacteria and a major player in the RNA-based regulation of gene expression, such as in the adaptive response to cell stress or in the induction of virulence (1, 2).Hfq was originally identified as a host factor in Escherichia coli for the replication of the Qβ phage (3), where it binds to the C-rich 3′ end of plus-strand viral RNA (4). Subsequently, physiological roles of Hfq were described in the regulation of mRNA translation and in mRNA degradation, where it modulates the processing of RNA 3′ ends (1,(5)(6)(7)(8). The most prominent function of Hfq, however, is its interaction with small regulatory RNAs (sRNAs) that act in trans and that are differentially expressed under various metabolic and environmental conditions (9, 10). They globally regulate gene expression via base-pairing to frequently entire sets of partially complementary mRNAs (11,12). Hfq stabilizes sRNAs in the absence of their targets (13). Hfq was also found to facilitate base-pairing to the mRNAs and help trigger subsequent steps, such as the repression of translation and/or the acceleration of decay, but also mRNA activation (11,14). Despite their structural diversity, the recognition of many sRNAs by Hfq is highly specific and even works across species barriers (15). It is an intriguing question how this selectivity is achieved.Crystal structures reveal that bacterial Hfq adopts an (L)Sm fold and forms homohexameric rings, whereas related (L)Sm proteins [Sm proteins and Sm-like (LSm) proteins] in archaea and eukaryotes are found to form homomeric or heteromeric heptamers, respectively (16). Two distinct RNA-binding sites have been described on opposite f...

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The structures of mutant forms of Hfq fromPseudomonas aeruginosareveal the importance of the conserved His57 for the protein hexamer organization

Cited by 25 publications

References 31 publications

Hexamer to Monomer Equilibrium of E. coli Hfq in Solution and Its Impact on RNA Annealing

Hexamer to Monomer Equilibrium of E. coli Hfq in Solution and Its Impact on RNA Annealing

Cell-Based Assay To Identify Inhibitors of the Hfq-sRNA Regulatory Pathway

Structural basis for RNA 3′-end recognition by Hfq

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