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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

Insertion within WD repeat 2.(a) The insertion (red) forms an important protrusion on the bottom of the domain. (b) Position of the insertion (red) in the context of the second blade only. Residues corresponding to WD repeat 2 are represented in light blue and the strand D of WD repeat 3 is shown in dark blue. (c) Comparison of blades 1 and 2 of WDR5 from H. sapiens (PDB:4CY2; pink) and Erb1 (blue). Side chains of conserved tryptophan corresponding to the strand C (in canonical WD repeats) are shown for both proteins. Black arrows indicate the position of 2D-2A loops. The letters in (b) and (c) indicate the position of each strand.
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pone.0123463.g004: Insertion within WD repeat 2.(a) The insertion (red) forms an important protrusion on the bottom of the domain. (b) Position of the insertion (red) in the context of the second blade only. Residues corresponding to WD repeat 2 are represented in light blue and the strand D of WD repeat 3 is shown in dark blue. (c) Comparison of blades 1 and 2 of WDR5 from H. sapiens (PDB:4CY2; pink) and Erb1 (blue). Side chains of conserved tryptophan corresponding to the strand C (in canonical WD repeats) are shown for both proteins. Black arrows indicate the position of 2D-2A loops. The letters in (b) and (c) indicate the position of each strand.

Mentions: Undoubtedly, the most distinctive feature of the core of the β-propeller in this study is the blade 2 which due to an insertion contains five, and not four, β-strands and shows a protrusion attributed to two α-helices (H2 and H3 on Fig 1c). Electron density map allowed us to trace and build model for residues 515–534 and 571–594, being the rest of the insertion unmodeled. This missing part seems to be Fungi specific as it becomes much shorter in higher eukaryotes (Fig 1c). Helix H2 (residues Tyr520-Asp532) appears between strands 2C and 2E and is attached to the base of the propeller (Fig 4a). In general, the sequence of the helix is poorly conserved, but it contains two invariable non-polar residues: Ile522 that makes hydrophobic contacts with the backbone of 2A-2B loop and Ile530 which interacts with the C-terminal fragment of strand 3D. The β-sheet corresponding to this blade is formed by strands 2A, 2B, 2C, 3D and 2E clearly indicating an alteration of a standard WD40 pattern (Fig 4b). Whereas strand 3D unambiguously indicates the beginning of WD3, the sequence of WD repeat 2 does not show any significant conservation but still contains strategic residues that allow formation of hydrophobic and electrostatic interactions with neighboring blades. Initial sequence-based analysis suggested that between WD repeats 2 and 3 there was an approximately 80-residue long segment which did not contain any WD pattern. Surprisingly, when we aligned the sequence of Ct domain of Erb1 with other non-Erb1/Bop1 β-propeller-containing proteins we could clearly see that Trp575 from strand 2E corresponded to Trp residue from WD dipeptide that typically appears in strand C (as in human WDR5 protein, PDB: 2H14) (Fig 4c) [37]. This fully conserved residue establishes important hydrophobic interactions with Ile592 from strand 2D and His629 located in 3D that are likely to be required for a proper conformation and attachment of the insertion to the side of the blade 2. We conclude that from an evolutionary point of view strand E corresponds to strand C from a canonical blade although displaced, in the second blade of Erb1, by a segment containing 2C-loop-H2. This insertion produced an important reorganization of the whole blade, altering the position and function of Trp-Asp dipeptide (Trp-Asn in this case). As a result, the second blade lacks the important Trp residue at the end of strand 2C that would guarantee correct approach between blades 1 and 2. We observe that in this case there is a different interaction network, conserved in Erb1/Bop1 family but not in other WD repeat-containing proteins, that involves strand 2D from blade 1 and a short α-helix, H3, from blade 2 (Gln580-Lys585). This helix inserts between strands 2E and 3D and possesses two non-conserved lysine residues (Lys581; Lys585) that interact with loop 2D-2A through hydrogen bonds. In consequence of this arrangement, α-helix H3 forms a lid that orientates close to a very hydrophobic area in blade 2 created by a segment of well conserved polar residues from strand 2B. It is important to keep in mind that loop 2D-2A is quite flexible and its vertical orientation makes the whole interface between blades 1 and 2 more opened when compared with the gaps between other blades which are completely covered by D-A loops (Fig 4c).


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)

Insertion within WD repeat 2.(a) The insertion (red) forms an important protrusion on the bottom of the domain. (b) Position of the insertion (red) in the context of the second blade only. Residues corresponding to WD repeat 2 are represented in light blue and the strand D of WD repeat 3 is shown in dark blue. (c) Comparison of blades 1 and 2 of WDR5 from H. sapiens (PDB:4CY2; pink) and Erb1 (blue). Side chains of conserved tryptophan corresponding to the strand C (in canonical WD repeats) are shown for both proteins. Black arrows indicate the position of 2D-2A loops. The letters in (b) and (c) indicate the position of each strand.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123463.g004: Insertion within WD repeat 2.(a) The insertion (red) forms an important protrusion on the bottom of the domain. (b) Position of the insertion (red) in the context of the second blade only. Residues corresponding to WD repeat 2 are represented in light blue and the strand D of WD repeat 3 is shown in dark blue. (c) Comparison of blades 1 and 2 of WDR5 from H. sapiens (PDB:4CY2; pink) and Erb1 (blue). Side chains of conserved tryptophan corresponding to the strand C (in canonical WD repeats) are shown for both proteins. Black arrows indicate the position of 2D-2A loops. The letters in (b) and (c) indicate the position of each strand.
Mentions: Undoubtedly, the most distinctive feature of the core of the β-propeller in this study is the blade 2 which due to an insertion contains five, and not four, β-strands and shows a protrusion attributed to two α-helices (H2 and H3 on Fig 1c). Electron density map allowed us to trace and build model for residues 515–534 and 571–594, being the rest of the insertion unmodeled. This missing part seems to be Fungi specific as it becomes much shorter in higher eukaryotes (Fig 1c). Helix H2 (residues Tyr520-Asp532) appears between strands 2C and 2E and is attached to the base of the propeller (Fig 4a). In general, the sequence of the helix is poorly conserved, but it contains two invariable non-polar residues: Ile522 that makes hydrophobic contacts with the backbone of 2A-2B loop and Ile530 which interacts with the C-terminal fragment of strand 3D. The β-sheet corresponding to this blade is formed by strands 2A, 2B, 2C, 3D and 2E clearly indicating an alteration of a standard WD40 pattern (Fig 4b). Whereas strand 3D unambiguously indicates the beginning of WD3, the sequence of WD repeat 2 does not show any significant conservation but still contains strategic residues that allow formation of hydrophobic and electrostatic interactions with neighboring blades. Initial sequence-based analysis suggested that between WD repeats 2 and 3 there was an approximately 80-residue long segment which did not contain any WD pattern. Surprisingly, when we aligned the sequence of Ct domain of Erb1 with other non-Erb1/Bop1 β-propeller-containing proteins we could clearly see that Trp575 from strand 2E corresponded to Trp residue from WD dipeptide that typically appears in strand C (as in human WDR5 protein, PDB: 2H14) (Fig 4c) [37]. This fully conserved residue establishes important hydrophobic interactions with Ile592 from strand 2D and His629 located in 3D that are likely to be required for a proper conformation and attachment of the insertion to the side of the blade 2. We conclude that from an evolutionary point of view strand E corresponds to strand C from a canonical blade although displaced, in the second blade of Erb1, by a segment containing 2C-loop-H2. This insertion produced an important reorganization of the whole blade, altering the position and function of Trp-Asp dipeptide (Trp-Asn in this case). As a result, the second blade lacks the important Trp residue at the end of strand 2C that would guarantee correct approach between blades 1 and 2. We observe that in this case there is a different interaction network, conserved in Erb1/Bop1 family but not in other WD repeat-containing proteins, that involves strand 2D from blade 1 and a short α-helix, H3, from blade 2 (Gln580-Lys585). This helix inserts between strands 2E and 3D and possesses two non-conserved lysine residues (Lys581; Lys585) that interact with loop 2D-2A through hydrogen bonds. In consequence of this arrangement, α-helix H3 forms a lid that orientates close to a very hydrophobic area in blade 2 created by a segment of well conserved polar residues from strand 2B. It is important to keep in mind that loop 2D-2A is quite flexible and its vertical orientation makes the whole interface between blades 1 and 2 more opened when compared with the gaps between other blades which are completely covered by D-A loops (Fig 4c).

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