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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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Interaction between KH2 and KH3 in the two- and four- (KH1–KH4) domain constructs. (A) Mapping of the residues of KH2 and KH3 whose 15N and 1H amide chemical shift changes in the two domain construct on a space filling representation of KH23. Residues of KH2 (233–305) and KH3 (317–418) whose amide resonances Δδavg (ppm) is <0.2 are in light grey, while residues whose Δδavg ≥ 0.2 are in orange for KH2 and magenta for KH3. Residues in the linker are in green, unassigned residues in dark grey. (B) The KH2–KH3 arrangement is conserved in KH1234 Superimposition of 15N-HSQC spectra of KH23 (233–418), green, and KSRP KH1234, purple. Grey labels correspond to KH2 signals which shift upon addition of the KH2–KH3 linker and/or KH3 binding. The absence of significant chemical shift changes in the resonances of KH2, KH3 and the linker indicate that the relative orientation of the KH2 and KH3 domains and their contacts with the linker are conserved in the longer protein.
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Figure 2: Interaction between KH2 and KH3 in the two- and four- (KH1–KH4) domain constructs. (A) Mapping of the residues of KH2 and KH3 whose 15N and 1H amide chemical shift changes in the two domain construct on a space filling representation of KH23. Residues of KH2 (233–305) and KH3 (317–418) whose amide resonances Δδavg (ppm) is <0.2 are in light grey, while residues whose Δδavg ≥ 0.2 are in orange for KH2 and magenta for KH3. Residues in the linker are in green, unassigned residues in dark grey. (B) The KH2–KH3 arrangement is conserved in KH1234 Superimposition of 15N-HSQC spectra of KH23 (233–418), green, and KSRP KH1234, purple. Grey labels correspond to KH2 signals which shift upon addition of the KH2–KH3 linker and/or KH3 binding. The absence of significant chemical shift changes in the resonances of KH2, KH3 and the linker indicate that the relative orientation of the KH2 and KH3 domains and their contacts with the linker are conserved in the longer protein.

Mentions: The superposition of the 15N-HSQC spectra (Supplementary Figure S1) of the isolated KH2 (233–305) and KH3 (317–418) domains on that of the two-domain KH23 construct (233–418) shows significant chemical shift differences (Δδavg > 0.02 ppm) for resonances of both domains which are mapped in Figure 2A. The largest chemical shift changes are observed for residues of KH2’s α1/α3 and KH3’s β1 (see the structure of KH2 in the next paragraph and ref. 6), indicating that the linker is likely to be sandwiched between the two domains. Indeed, the addition of the linker to the KH2 domain (KH2+linker construct) results in very substantial chemical shift changes in the KH2 resonances (Supplementary Figure S1), while inclusion of KH3 causes only small additional chemical shift perturbations. Furthermore, resonances of the last seven amino acids of the linker (317–324), which are not visible when this short stretch is attached to KH3 in the (317–418) construct, become clearly visible in the KH23 construct, indicating that further contacts are taking place in the full two-domain construct. Importantly, no significant chemical shift differences are observed between the isolated KH23 and the two domains within the KH1234 construct (Figure 2B), which confirms that the features of the KH23 interaction are conserved in the longer protein.Figure 2.


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)

Interaction between KH2 and KH3 in the two- and four- (KH1–KH4) domain constructs. (A) Mapping of the residues of KH2 and KH3 whose 15N and 1H amide chemical shift changes in the two domain construct on a space filling representation of KH23. Residues of KH2 (233–305) and KH3 (317–418) whose amide resonances Δδavg (ppm) is <0.2 are in light grey, while residues whose Δδavg ≥ 0.2 are in orange for KH2 and magenta for KH3. Residues in the linker are in green, unassigned residues in dark grey. (B) The KH2–KH3 arrangement is conserved in KH1234 Superimposition of 15N-HSQC spectra of KH23 (233–418), green, and KSRP KH1234, purple. Grey labels correspond to KH2 signals which shift upon addition of the KH2–KH3 linker and/or KH3 binding. The absence of significant chemical shift changes in the resonances of KH2, KH3 and the linker indicate that the relative orientation of the KH2 and KH3 domains and their contacts with the linker are conserved in the longer protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2926597&req=5

Figure 2: Interaction between KH2 and KH3 in the two- and four- (KH1–KH4) domain constructs. (A) Mapping of the residues of KH2 and KH3 whose 15N and 1H amide chemical shift changes in the two domain construct on a space filling representation of KH23. Residues of KH2 (233–305) and KH3 (317–418) whose amide resonances Δδavg (ppm) is <0.2 are in light grey, while residues whose Δδavg ≥ 0.2 are in orange for KH2 and magenta for KH3. Residues in the linker are in green, unassigned residues in dark grey. (B) The KH2–KH3 arrangement is conserved in KH1234 Superimposition of 15N-HSQC spectra of KH23 (233–418), green, and KSRP KH1234, purple. Grey labels correspond to KH2 signals which shift upon addition of the KH2–KH3 linker and/or KH3 binding. The absence of significant chemical shift changes in the resonances of KH2, KH3 and the linker indicate that the relative orientation of the KH2 and KH3 domains and their contacts with the linker are conserved in the longer protein.
Mentions: The superposition of the 15N-HSQC spectra (Supplementary Figure S1) of the isolated KH2 (233–305) and KH3 (317–418) domains on that of the two-domain KH23 construct (233–418) shows significant chemical shift differences (Δδavg > 0.02 ppm) for resonances of both domains which are mapped in Figure 2A. The largest chemical shift changes are observed for residues of KH2’s α1/α3 and KH3’s β1 (see the structure of KH2 in the next paragraph and ref. 6), indicating that the linker is likely to be sandwiched between the two domains. Indeed, the addition of the linker to the KH2 domain (KH2+linker construct) results in very substantial chemical shift changes in the KH2 resonances (Supplementary Figure S1), while inclusion of KH3 causes only small additional chemical shift perturbations. Furthermore, resonances of the last seven amino acids of the linker (317–324), which are not visible when this short stretch is attached to KH3 in the (317–418) construct, become clearly visible in the KH23 construct, indicating that further contacts are taking place in the full two-domain construct. Importantly, no significant chemical shift differences are observed between the isolated KH23 and the two domains within the KH1234 construct (Figure 2B), which confirms that the features of the KH23 interaction are conserved in the longer protein.Figure 2.

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