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Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha.

Weber J, Bao H, Hartlmüller C, Wang Z, Windhager A, Janowski R, Madl T, Jin P, Niessing D - Elife (2016)

Bottom Line: It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome.Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function.By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany.

ABSTRACT
The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus

1H,15N HSQC NMR spectra showing NMR titrations of 15N-labeled Pur-alpha repeat I-II (50 µM) with increasing amounts of unlabeled GCGGA ssDNA and RNA, respectively. (A) Titration with DNA. Peaks corresponding to the free and DNA-bound protein states (protein:DNA 1:0, 1:0.5, 1:1, 1:2.5 and 1:5 ratio) are represented in blue, cyan, green, orange and red, respectively. (B) Titration with RNA. Peaks corresponding to the free and RNA-bound protein states (protein:RNA 1:0, 1:0.5, 1:1, 1:2.5, and 1:5 ratio) are represented with the same color code as in (A).DOI:http://dx.doi.org/10.7554/eLife.11297.005
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fig1s2: 1H,15N HSQC NMR spectra showing NMR titrations of 15N-labeled Pur-alpha repeat I-II (50 µM) with increasing amounts of unlabeled GCGGA ssDNA and RNA, respectively. (A) Titration with DNA. Peaks corresponding to the free and DNA-bound protein states (protein:DNA 1:0, 1:0.5, 1:1, 1:2.5 and 1:5 ratio) are represented in blue, cyan, green, orange and red, respectively. (B) Titration with RNA. Peaks corresponding to the free and RNA-bound protein states (protein:RNA 1:0, 1:0.5, 1:1, 1:2.5, and 1:5 ratio) are represented with the same color code as in (A).DOI:http://dx.doi.org/10.7554/eLife.11297.005

Mentions: For a more comprehensive, residue-resolved comparison of ssDNA and ssRNA binding, we performed NMR chemical shift titration experiments with 15N-labeled Drosophila Pur-alpha repeat I-II (Figure 1—figure supplement 1C) and short unlabeled GCGGA (5 nt) DNA and RNA fragments. The 1H,15N HSQC NMR spectrum of Pur-alpha alone shows well separated cross peaks (Figure 1E; Figure 1—figure supplement 2A, B), indicating that the protein is correctly folded. Addition of either ssDNA or ssRNA resulted in almost identical, well-localized chemical shift perturbations of backbone and sidechain amide protons (Figure 1E; Figure 1—figure supplement 2A, B). Most NMR signals of residues involved in binding disappeared upon addition of DNA/RNA, thus pointing toward an intermediate exchange regime, which is characteristic for binding affinities in the high nanomolar to micromolar range. In summary, the NMR titration experiments indicate identical binding modes of PUR repeat I-II for ssDNA and for ssRNA involving the same residues in both cases.


Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha.

Weber J, Bao H, Hartlmüller C, Wang Z, Windhager A, Janowski R, Madl T, Jin P, Niessing D - Elife (2016)

1H,15N HSQC NMR spectra showing NMR titrations of 15N-labeled Pur-alpha repeat I-II (50 µM) with increasing amounts of unlabeled GCGGA ssDNA and RNA, respectively. (A) Titration with DNA. Peaks corresponding to the free and DNA-bound protein states (protein:DNA 1:0, 1:0.5, 1:1, 1:2.5 and 1:5 ratio) are represented in blue, cyan, green, orange and red, respectively. (B) Titration with RNA. Peaks corresponding to the free and RNA-bound protein states (protein:RNA 1:0, 1:0.5, 1:1, 1:2.5, and 1:5 ratio) are represented with the same color code as in (A).DOI:http://dx.doi.org/10.7554/eLife.11297.005
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4764581&req=5

fig1s2: 1H,15N HSQC NMR spectra showing NMR titrations of 15N-labeled Pur-alpha repeat I-II (50 µM) with increasing amounts of unlabeled GCGGA ssDNA and RNA, respectively. (A) Titration with DNA. Peaks corresponding to the free and DNA-bound protein states (protein:DNA 1:0, 1:0.5, 1:1, 1:2.5 and 1:5 ratio) are represented in blue, cyan, green, orange and red, respectively. (B) Titration with RNA. Peaks corresponding to the free and RNA-bound protein states (protein:RNA 1:0, 1:0.5, 1:1, 1:2.5, and 1:5 ratio) are represented with the same color code as in (A).DOI:http://dx.doi.org/10.7554/eLife.11297.005
Mentions: For a more comprehensive, residue-resolved comparison of ssDNA and ssRNA binding, we performed NMR chemical shift titration experiments with 15N-labeled Drosophila Pur-alpha repeat I-II (Figure 1—figure supplement 1C) and short unlabeled GCGGA (5 nt) DNA and RNA fragments. The 1H,15N HSQC NMR spectrum of Pur-alpha alone shows well separated cross peaks (Figure 1E; Figure 1—figure supplement 2A, B), indicating that the protein is correctly folded. Addition of either ssDNA or ssRNA resulted in almost identical, well-localized chemical shift perturbations of backbone and sidechain amide protons (Figure 1E; Figure 1—figure supplement 2A, B). Most NMR signals of residues involved in binding disappeared upon addition of DNA/RNA, thus pointing toward an intermediate exchange regime, which is characteristic for binding affinities in the high nanomolar to micromolar range. In summary, the NMR titration experiments indicate identical binding modes of PUR repeat I-II for ssDNA and for ssRNA involving the same residues in both cases.

Bottom Line: It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome.Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function.By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany.

ABSTRACT
The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus