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The carboxy-terminal domain of Erb1 is a seven-bladed ß-propeller that binds RNA.

Wegrecki M, Marcin W, Neira JL, Bravo J - PLoS ONE (2015)

Bottom Line: This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat.The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding.Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, c/ Jaime Roig 11, 46010 Valencia, Spain.

ABSTRACT
Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other β-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

No MeSH data available.


Related in: MedlinePlus

Erb1 degradation and β-propeller general features.(a) Coomassie—stained SDS-PAGE showing a severe degradation pattern of full-length Erb1 when incubated at 4°C overnight. The N-terminal and C-terminal degradation products are marked. MW in kDa is shown next to the ladder. (b) Ribbon representation of the β-propeller domain of Erb1 seen from the top (by convention the top face is described as the one that contains loops B-C and D-A). The blades numbering is counterclockwise and the β-strands nomenclature follows as shown for blade 4. Black arrow indicates the Velcro-like closure of the domain. (c) Sequence multi alignment of the carboxy-terminal domain of Erb1/Bop1 from Saccharomyces cerevisiae (Sc), Chaetomium thermophilum (Cht), Homo sapiens (Hs), Mus musculus (Mm) and Danio rerio (Dr). For clarity only the residues present in the final pdb model are shown. Conserved amino acids are marked with shadows. Secondary structure assignment is shown on the top of the alignment. Numbers of β-strands correspond to the WD repeats of the protein. Red rectangles mark conserved Asp in B-C loops and red squares indicate residues forming His-Thr/Ser-Asp triads. Green rectangles show basic residues that form a putative RNA-binding site.
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pone.0123463.g001: Erb1 degradation and β-propeller general features.(a) Coomassie—stained SDS-PAGE showing a severe degradation pattern of full-length Erb1 when incubated at 4°C overnight. The N-terminal and C-terminal degradation products are marked. MW in kDa is shown next to the ladder. (b) Ribbon representation of the β-propeller domain of Erb1 seen from the top (by convention the top face is described as the one that contains loops B-C and D-A). The blades numbering is counterclockwise and the β-strands nomenclature follows as shown for blade 4. Black arrow indicates the Velcro-like closure of the domain. (c) Sequence multi alignment of the carboxy-terminal domain of Erb1/Bop1 from Saccharomyces cerevisiae (Sc), Chaetomium thermophilum (Cht), Homo sapiens (Hs), Mus musculus (Mm) and Danio rerio (Dr). For clarity only the residues present in the final pdb model are shown. Conserved amino acids are marked with shadows. Secondary structure assignment is shown on the top of the alignment. Numbers of β-strands correspond to the WD repeats of the protein. Red rectangles mark conserved Asp in B-C loops and red squares indicate residues forming His-Thr/Ser-Asp triads. Green rectangles show basic residues that form a putative RNA-binding site.

Mentions: In order to confirm if the crystals from initial screenings contained Nop7 or a fragment of Erb1, 15 crystals were analyzed by SDS-PAGE and, after staining with Coomassie Blue, only a single faint band of approximately 45 kDa could be observed on the gel. When the stability of Nop7, Erb1 and the Nop7/Erb1 complex was assayed, it was seen that after 24h of incubation at 4°C Erb1 started to show a clear pattern of degradation that was visible even upon binding to Nop7 and led to the apparition of the 45kDa band as seen for the crystals (Fig 1a). The mass-spec analysis confirmed that the lower MW band corresponded to the C-terminal region of Erb1 and contained the whole β-propeller domain of the protein.


The carboxy-terminal domain of Erb1 is a seven-bladed ß-propeller that binds RNA.

Wegrecki M, Marcin W, Neira JL, Bravo J - PLoS ONE (2015)

Erb1 degradation and β-propeller general features.(a) Coomassie—stained SDS-PAGE showing a severe degradation pattern of full-length Erb1 when incubated at 4°C overnight. The N-terminal and C-terminal degradation products are marked. MW in kDa is shown next to the ladder. (b) Ribbon representation of the β-propeller domain of Erb1 seen from the top (by convention the top face is described as the one that contains loops B-C and D-A). The blades numbering is counterclockwise and the β-strands nomenclature follows as shown for blade 4. Black arrow indicates the Velcro-like closure of the domain. (c) Sequence multi alignment of the carboxy-terminal domain of Erb1/Bop1 from Saccharomyces cerevisiae (Sc), Chaetomium thermophilum (Cht), Homo sapiens (Hs), Mus musculus (Mm) and Danio rerio (Dr). For clarity only the residues present in the final pdb model are shown. Conserved amino acids are marked with shadows. Secondary structure assignment is shown on the top of the alignment. Numbers of β-strands correspond to the WD repeats of the protein. Red rectangles mark conserved Asp in B-C loops and red squares indicate residues forming His-Thr/Ser-Asp triads. Green rectangles show basic residues that form a putative RNA-binding site.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400149&req=5

pone.0123463.g001: Erb1 degradation and β-propeller general features.(a) Coomassie—stained SDS-PAGE showing a severe degradation pattern of full-length Erb1 when incubated at 4°C overnight. The N-terminal and C-terminal degradation products are marked. MW in kDa is shown next to the ladder. (b) Ribbon representation of the β-propeller domain of Erb1 seen from the top (by convention the top face is described as the one that contains loops B-C and D-A). The blades numbering is counterclockwise and the β-strands nomenclature follows as shown for blade 4. Black arrow indicates the Velcro-like closure of the domain. (c) Sequence multi alignment of the carboxy-terminal domain of Erb1/Bop1 from Saccharomyces cerevisiae (Sc), Chaetomium thermophilum (Cht), Homo sapiens (Hs), Mus musculus (Mm) and Danio rerio (Dr). For clarity only the residues present in the final pdb model are shown. Conserved amino acids are marked with shadows. Secondary structure assignment is shown on the top of the alignment. Numbers of β-strands correspond to the WD repeats of the protein. Red rectangles mark conserved Asp in B-C loops and red squares indicate residues forming His-Thr/Ser-Asp triads. Green rectangles show basic residues that form a putative RNA-binding site.
Mentions: In order to confirm if the crystals from initial screenings contained Nop7 or a fragment of Erb1, 15 crystals were analyzed by SDS-PAGE and, after staining with Coomassie Blue, only a single faint band of approximately 45 kDa could be observed on the gel. When the stability of Nop7, Erb1 and the Nop7/Erb1 complex was assayed, it was seen that after 24h of incubation at 4°C Erb1 started to show a clear pattern of degradation that was visible even upon binding to Nop7 and led to the apparition of the 45kDa band as seen for the crystals (Fig 1a). The mass-spec analysis confirmed that the lower MW band corresponded to the C-terminal region of Erb1 and contained the whole β-propeller domain of the protein.

Bottom Line: This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat.The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding.Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, c/ Jaime Roig 11, 46010 Valencia, Spain.

ABSTRACT
Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other β-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

No MeSH data available.


Related in: MedlinePlus