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Orientation of the central domains of KSRP and its implications for the interaction with the RNA targets.

Díaz-Moreno I, Hollingworth D, Kelly G, Martin S, García-Mayoral M, Briata P, Gherzi R, Ramos A - Nucleic Acids Res. (2010)

Bottom Line: KSRP is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for cellular proliferation and inflammatory response factors.The selectivity of this mRNA degradation mechanism relies on KSRP recognition of AU-rich elements in the mRNA 3'UTR, that is mediated by KSRP's KH domains.We also show that this inter-domain arrangement is important to the interaction with KSRP's RNA targets.

View Article: PubMed Central - PubMed

Affiliation: Molecular Structure Division, MRC National Institute for Medical Research, The Ridgeway, London, UK.

ABSTRACT
KSRP is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for cellular proliferation and inflammatory response factors. The selectivity of this mRNA degradation mechanism relies on KSRP recognition of AU-rich elements in the mRNA 3'UTR, that is mediated by KSRP's KH domains. Our structural analysis shows that the inter-domain linker orients the two central KH domains of KSRP-and their RNA-binding surfaces-creating a two-domain unit. We also show that this inter-domain arrangement is important to the interaction with KSRP's RNA targets.

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Internal motions in KH2, KH3 and KH23. T2 (A) and Heteronuclear NOE (B) values of KH2 (233–305) and KH3 (317–418) amide resonances, in grey (as reported in 8 and 6, respectively), are compared with equivalent data on KH23 (233–418) in black. The positions of the variable loop(s) and inter-domain linker in the protein sequence are indicated at the bottom of the figure. The arrows span the KH2 and KH3 domains and two vertical lines define the boundaries of the more and less flexible parts of the linker. Residues in the invariant loops of KH2 and KH3 are characterized by T2 and heteronuclear values shorter than the average, suggesting conformational exchanges with rates near µs to ms time scale (30) while residues in the variable loops displays a behaviour consistent with motions in the nanosecond range.
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Figure 3: Internal motions in KH2, KH3 and KH23. T2 (A) and Heteronuclear NOE (B) values of KH2 (233–305) and KH3 (317–418) amide resonances, in grey (as reported in 8 and 6, respectively), are compared with equivalent data on KH23 (233–418) in black. The positions of the variable loop(s) and inter-domain linker in the protein sequence are indicated at the bottom of the figure. The arrows span the KH2 and KH3 domains and two vertical lines define the boundaries of the more and less flexible parts of the linker. Residues in the invariant loops of KH2 and KH3 are characterized by T2 and heteronuclear values shorter than the average, suggesting conformational exchanges with rates near µs to ms time scale (30) while residues in the variable loops displays a behaviour consistent with motions in the nanosecond range.

Mentions: 15N T1, T2, and heteronuclear NOE values recorded on the KH2, KH3 and KH23 proteins indicate that KH23 encompasses two relatively rigid regions (corresponding to the two KH domains) joined by a more mobile 18-residue long linker (Figure 3 and Supplementary Figure S2). The intra-domain trends of 15N T1, T2 and heteronuclear NOE values for KH2 and KH3 are consistent with what is observed for other KH domains and do not change from the single to the di-domain constructs (Figure 3). The linker comprises two distinct regions corresponding to amino acids 306–310 and 311–324. The trend and values of the relaxation parameters of amino acids 306–310 are consistent with a fully flexible protein chain (30), while the ones of residues 311–324 are in between those of the structured KH domains and those of the flexible 306–310 residues (Figure 3). This second part of the linker is much more flexible in the KH2+linker construct (Supplementary Figure S2) in agreement with the linker being sandwiched between the two KH domains. The KH23 construct has a rotational correlation time (τc) of 8.92 ns, in the range of τc/MW reported by Dayie and co-workers (31) and significantly larger than the one of the isolated KH2 and KH3 domains (5.27 and 6.36 ns in the isolated domains), consistently with the two domains interacting.Figure 3.


Orientation of the central domains of KSRP and its implications for the interaction with the RNA targets.

Díaz-Moreno I, Hollingworth D, Kelly G, Martin S, García-Mayoral M, Briata P, Gherzi R, Ramos A - Nucleic Acids Res. (2010)

Internal motions in KH2, KH3 and KH23. T2 (A) and Heteronuclear NOE (B) values of KH2 (233–305) and KH3 (317–418) amide resonances, in grey (as reported in 8 and 6, respectively), are compared with equivalent data on KH23 (233–418) in black. The positions of the variable loop(s) and inter-domain linker in the protein sequence are indicated at the bottom of the figure. The arrows span the KH2 and KH3 domains and two vertical lines define the boundaries of the more and less flexible parts of the linker. Residues in the invariant loops of KH2 and KH3 are characterized by T2 and heteronuclear values shorter than the average, suggesting conformational exchanges with rates near µs to ms time scale (30) while residues in the variable loops displays a behaviour consistent with motions in the nanosecond range.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926597&req=5

Figure 3: Internal motions in KH2, KH3 and KH23. T2 (A) and Heteronuclear NOE (B) values of KH2 (233–305) and KH3 (317–418) amide resonances, in grey (as reported in 8 and 6, respectively), are compared with equivalent data on KH23 (233–418) in black. The positions of the variable loop(s) and inter-domain linker in the protein sequence are indicated at the bottom of the figure. The arrows span the KH2 and KH3 domains and two vertical lines define the boundaries of the more and less flexible parts of the linker. Residues in the invariant loops of KH2 and KH3 are characterized by T2 and heteronuclear values shorter than the average, suggesting conformational exchanges with rates near µs to ms time scale (30) while residues in the variable loops displays a behaviour consistent with motions in the nanosecond range.
Mentions: 15N T1, T2, and heteronuclear NOE values recorded on the KH2, KH3 and KH23 proteins indicate that KH23 encompasses two relatively rigid regions (corresponding to the two KH domains) joined by a more mobile 18-residue long linker (Figure 3 and Supplementary Figure S2). The intra-domain trends of 15N T1, T2 and heteronuclear NOE values for KH2 and KH3 are consistent with what is observed for other KH domains and do not change from the single to the di-domain constructs (Figure 3). The linker comprises two distinct regions corresponding to amino acids 306–310 and 311–324. The trend and values of the relaxation parameters of amino acids 306–310 are consistent with a fully flexible protein chain (30), while the ones of residues 311–324 are in between those of the structured KH domains and those of the flexible 306–310 residues (Figure 3). This second part of the linker is much more flexible in the KH2+linker construct (Supplementary Figure S2) in agreement with the linker being sandwiched between the two KH domains. The KH23 construct has a rotational correlation time (τc) of 8.92 ns, in the range of τc/MW reported by Dayie and co-workers (31) and significantly larger than the one of the isolated KH2 and KH3 domains (5.27 and 6.36 ns in the isolated domains), consistently with the two domains interacting.Figure 3.

Bottom Line: KSRP is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for cellular proliferation and inflammatory response factors.The selectivity of this mRNA degradation mechanism relies on KSRP recognition of AU-rich elements in the mRNA 3'UTR, that is mediated by KSRP's KH domains.We also show that this inter-domain arrangement is important to the interaction with KSRP's RNA targets.

View Article: PubMed Central - PubMed

Affiliation: Molecular Structure Division, MRC National Institute for Medical Research, The Ridgeway, London, UK.

ABSTRACT
KSRP is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for cellular proliferation and inflammatory response factors. The selectivity of this mRNA degradation mechanism relies on KSRP recognition of AU-rich elements in the mRNA 3'UTR, that is mediated by KSRP's KH domains. Our structural analysis shows that the inter-domain linker orients the two central KH domains of KSRP-and their RNA-binding surfaces-creating a two-domain unit. We also show that this inter-domain arrangement is important to the interaction with KSRP's RNA targets.

Show MeSH
Related in: MedlinePlus