Limits...
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.


Erb1 insertion is disordered in solution.(a) Fluorescence spectrum of Erb1518-586 obtained by excitation at 280 nm. (b) Far-UV CD spectrum of Erb1518-586. (c) Amide (left) and alkyl (right) regions of the 1D-1H- NMR spectrum of Erb1518-586. (d)15N-1H HSQC spectrum of Erb1518-586.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400149&req=5

pone.0123463.g005: Erb1 insertion is disordered in solution.(a) Fluorescence spectrum of Erb1518-586 obtained by excitation at 280 nm. (b) Far-UV CD spectrum of Erb1518-586. (c) Amide (left) and alkyl (right) regions of the 1D-1H- NMR spectrum of Erb1518-586. (d)15N-1H HSQC spectrum of Erb1518-586.

Mentions: We studied the conformational propensities of the isolated insertion, the fragment comprising residues Tyr518-Asp586 of Erb1, in solution. Fluorescence spectrum showed the maximum at 345 nm, suggesting that the sole Trp, Trp575, was highly solvent-exposed (Fig 5a). Furthermore, its far-UV CD spectrum had an intense minimum at 200 nm, and a shoulder at 222 nm (Fig 5b), which suggests the presence of helical or turn-like conformations. Those structured conformations, however, were not stable enough, since thermal denaturations followed by far-UV CD did not show any sigmoidal behavior (data not shown). The 1D-1H–NMR spectrum had all the amide protons clustered between 8.0 and 8.5 ppm. Moreover, the indole proton appeared at 10.19 ppm, close to the expected value of a random-coil indole moiety [38], and thus further supporting the fluorescence results. In the 1D-1H-NMR spectrum, there was no down-field shifted Hα protons (suggesting the absence of β-strands); however, there were a few up-field shifted methyl protons, which are probably close, in the primary structure, to some of the aromatic groups (Fig 5c). Finally, the 2D-15N-HSQC experiments (Fig 5d) had a very narrow dispersion in the amide proton region of 0.4 ppm that is centered around the random-coil chemical shift values for NHs (between 8.2 and 8.4 ppm), confirming the 1D-NMR results. In addition, there was a lower number of cross-peaks than we should expect (79); the absence of the peaks is either probably due to chemical exchange broadening or rapid amide exchange, as expected in natively unfolded proteins [39].


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 insertion is disordered in solution.(a) Fluorescence spectrum of Erb1518-586 obtained by excitation at 280 nm. (b) Far-UV CD spectrum of Erb1518-586. (c) Amide (left) and alkyl (right) regions of the 1D-1H- NMR spectrum of Erb1518-586. (d)15N-1H HSQC spectrum of Erb1518-586.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123463.g005: Erb1 insertion is disordered in solution.(a) Fluorescence spectrum of Erb1518-586 obtained by excitation at 280 nm. (b) Far-UV CD spectrum of Erb1518-586. (c) Amide (left) and alkyl (right) regions of the 1D-1H- NMR spectrum of Erb1518-586. (d)15N-1H HSQC spectrum of Erb1518-586.
Mentions: We studied the conformational propensities of the isolated insertion, the fragment comprising residues Tyr518-Asp586 of Erb1, in solution. Fluorescence spectrum showed the maximum at 345 nm, suggesting that the sole Trp, Trp575, was highly solvent-exposed (Fig 5a). Furthermore, its far-UV CD spectrum had an intense minimum at 200 nm, and a shoulder at 222 nm (Fig 5b), which suggests the presence of helical or turn-like conformations. Those structured conformations, however, were not stable enough, since thermal denaturations followed by far-UV CD did not show any sigmoidal behavior (data not shown). The 1D-1H–NMR spectrum had all the amide protons clustered between 8.0 and 8.5 ppm. Moreover, the indole proton appeared at 10.19 ppm, close to the expected value of a random-coil indole moiety [38], and thus further supporting the fluorescence results. In the 1D-1H-NMR spectrum, there was no down-field shifted Hα protons (suggesting the absence of β-strands); however, there were a few up-field shifted methyl protons, which are probably close, in the primary structure, to some of the aromatic groups (Fig 5c). Finally, the 2D-15N-HSQC experiments (Fig 5d) had a very narrow dispersion in the amide proton region of 0.4 ppm that is centered around the random-coil chemical shift values for NHs (between 8.2 and 8.4 ppm), confirming the 1D-NMR results. In addition, there was a lower number of cross-peaks than we should expect (79); the absence of the peaks is either probably due to chemical exchange broadening or rapid amide exchange, as expected in natively unfolded proteins [39].

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.