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Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq.

Beich-Frandsen M, Vecerek B, Konarev PV, Sjöblom B, Kloiber K, Hämmerle H, Rajkowitsch L, Miles AJ, Kontaxis G, Wallace BA, Svergun DI, Konrat R, Bläsi U, Djinovic-Carugo K - Nucleic Acids Res. (2011)

Bottom Line: These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions.We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments.This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria.

ABSTRACT
The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec).

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SRCD spectra of HfqEc (thick line), in complex with DsrA34 (open squares), and hfq126 RNA (thin line). The error bars shown represent one standard deviation between replicate scans.
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Figure 7: SRCD spectra of HfqEc (thick line), in complex with DsrA34 (open squares), and hfq126 RNA (thin line). The error bars shown represent one standard deviation between replicate scans.

Mentions: In order to monitor changes in the structure of the C-terminal extension of HfqEc in the presence of RNA, we next used SRCD spectroscopy. Both HfqEc65 and full-length HfqEc bind the sRNA DsrA (82) and also the Hfq binding fragment of DsrA (DsrA34; data not shown) with comparable affinity, indicating that the C-terminus is not required for binding of the sRNA. As the SRCD spectra of HfqEc alone and in the presence of DsrA34 were indistinguishable (Figure 7), the known binding mode of DsrA to the inner core of the HfqEc hexamer (29), apparently does not affect the structure of the protein. In contrast to sRNAs, longer (m)RNA fragments did not bind to the HfqEc65 but depended on the presence of the C-terminus (18). Interestingly a 126-nt long fragment of hfq mRNA, which failed to bind to HfqEc65 but bound to full-length HfqEc (18), produced significant increases in the 185–200 nm region only upon binding to full-length HfqEc, reflecting an increase in the amount of ordered (β-strand or α-helical) conformations for this part of Hfq. The increase is consistent with a combination of decrease of disordered and increase in ordered residues. Altogether this suggests that RNA binding leads to an ordering of the C-terminal extension of Hfq. Many intrinsically disordered proteins undergo transitions to more ordered states or fold into stable secondary or tertiary structures upon binding to their targets. They undergo coupled folding and binding, forming complexes with high specificity and relatively low affinity, which is critical for processes in which not only specific association but also subsequent dissociation of binding partners is required (87).Figure 7.


Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq.

Beich-Frandsen M, Vecerek B, Konarev PV, Sjöblom B, Kloiber K, Hämmerle H, Rajkowitsch L, Miles AJ, Kontaxis G, Wallace BA, Svergun DI, Konrat R, Bläsi U, Djinovic-Carugo K - Nucleic Acids Res. (2011)

SRCD spectra of HfqEc (thick line), in complex with DsrA34 (open squares), and hfq126 RNA (thin line). The error bars shown represent one standard deviation between replicate scans.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113564&req=5

Figure 7: SRCD spectra of HfqEc (thick line), in complex with DsrA34 (open squares), and hfq126 RNA (thin line). The error bars shown represent one standard deviation between replicate scans.
Mentions: In order to monitor changes in the structure of the C-terminal extension of HfqEc in the presence of RNA, we next used SRCD spectroscopy. Both HfqEc65 and full-length HfqEc bind the sRNA DsrA (82) and also the Hfq binding fragment of DsrA (DsrA34; data not shown) with comparable affinity, indicating that the C-terminus is not required for binding of the sRNA. As the SRCD spectra of HfqEc alone and in the presence of DsrA34 were indistinguishable (Figure 7), the known binding mode of DsrA to the inner core of the HfqEc hexamer (29), apparently does not affect the structure of the protein. In contrast to sRNAs, longer (m)RNA fragments did not bind to the HfqEc65 but depended on the presence of the C-terminus (18). Interestingly a 126-nt long fragment of hfq mRNA, which failed to bind to HfqEc65 but bound to full-length HfqEc (18), produced significant increases in the 185–200 nm region only upon binding to full-length HfqEc, reflecting an increase in the amount of ordered (β-strand or α-helical) conformations for this part of Hfq. The increase is consistent with a combination of decrease of disordered and increase in ordered residues. Altogether this suggests that RNA binding leads to an ordering of the C-terminal extension of Hfq. Many intrinsically disordered proteins undergo transitions to more ordered states or fold into stable secondary or tertiary structures upon binding to their targets. They undergo coupled folding and binding, forming complexes with high specificity and relatively low affinity, which is critical for processes in which not only specific association but also subsequent dissociation of binding partners is required (87).Figure 7.

Bottom Line: These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions.We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments.This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria.

ABSTRACT
The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec).

Show MeSH
Related in: MedlinePlus