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Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex.

Berke IC, Boehmer T, Blobel G, Schwartz TU - J. Cell Biol. (2004)

Bottom Line: We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller.Other beta-propellers are predicted in a third of all nucleoporins.These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only approximately 30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller. The surface properties and conservation of the Nup133 beta-propeller suggest it may mediate multiple interactions with other proteins. Other beta-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

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Structural alignment of the seven blades in the Nup133 β-propeller. (a and b) Superposition of 21 Cα positions from each blade calculated by the program MultiProt (http://bioinfo3d.cs.tau.ac.il/MultiProt/). Gray spheres mark the aligned Cα atoms from blade 3. Strand A lines the inner channel of the propeller and is roughly parallel to the pseudo-sevenfold axis. Blade 2 (orange), blade 5 (cyan), and blade 7 (purple) contain insertions. (c) Structure-based sequence alignment of Nup133 NTD blades. Conserved hydrophobic positions are shaded yellow. Clathrin NTD propeller blades are shown for comparison. The sequence is shown in Joy formatting (http://www-cryst.bioc.cam.ac.uk/~joy/). β-Strands are shown in blue, 310 helices are shown in maroon, capitalized residues are solvent inaccessible, bold indicates a sidechain-backbone amide H-bond, underline indicates sidechain-backbone carbonyl H-bond, and italics indicates a residue with positive phi values. Gray residues were not built in the model. Bracketed numbers refer to residues not depicted.
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fig3: Structural alignment of the seven blades in the Nup133 β-propeller. (a and b) Superposition of 21 Cα positions from each blade calculated by the program MultiProt (http://bioinfo3d.cs.tau.ac.il/MultiProt/). Gray spheres mark the aligned Cα atoms from blade 3. Strand A lines the inner channel of the propeller and is roughly parallel to the pseudo-sevenfold axis. Blade 2 (orange), blade 5 (cyan), and blade 7 (purple) contain insertions. (c) Structure-based sequence alignment of Nup133 NTD blades. Conserved hydrophobic positions are shaded yellow. Clathrin NTD propeller blades are shown for comparison. The sequence is shown in Joy formatting (http://www-cryst.bioc.cam.ac.uk/~joy/). β-Strands are shown in blue, 310 helices are shown in maroon, capitalized residues are solvent inaccessible, bold indicates a sidechain-backbone amide H-bond, underline indicates sidechain-backbone carbonyl H-bond, and italics indicates a residue with positive phi values. Gray residues were not built in the model. Bracketed numbers refer to residues not depicted.

Mentions: We expressed the NTD of hNup133 (residues 67–514) in E. coli, crystallized it, and solved the structure to 2.35 Å. hNup133 NTD is a β-propeller with seven, four-stranded β-sheets arranged face to face around a central water-filled cavity (Fig. 2, a and b; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200408109/DC1). The polypeptide chain enters each propeller blade from the innermost strand and folds in an antiparallel manner (Fig. 2 a and Fig. 3 a, strands labeled A–D). Blade 7 of the propeller consists of the innermost three strands from the COOH terminus with the blade completed by the NH2 terminus of the domain. This 3 + 1 molecular clasp architecture is a common feature for stabilizing β-propellers (Paoli, 2001). The repeating antiparallel structure results in a top surface composed of the loops connecting strand D of one blade to strand A of the next (DA loop) as well as the BC loop within each blade, whereas the bottom surface is composed of the AB and CD loops. Two significant α-helical insertions are present in DA loops (Fig. 2, a and b, pink). The α1 helix inserts between the interface of blades 7 and 1, displacing blade 7 away and blade 1 toward the central axis. Blade 5 is extended and curls around helix α2 located in the DA loop connecting blades 4 and 5. This helical “wing” juts out from the core of the propeller by 15 Å. A disordered 20-residue insertion is present in the DA loop connecting blades 3 and 4 (DA34). The propeller has an overall diameter of 45–50 Å and the β-sheet core is ∼25-Å thick. Blade 2 has a short 310 helix just before strand 2A that projects into the top of the inner channel (Fig. 2 a; and Fig. 3 a, orange) and strand 2A is oriented away from the pseudo-sevenfold axis such that the bottom of the channel is wider than the top. The inner channel is oval shaped, being 12–16 Å wide at the top and 12–20 Å at the bottom (Cα to Cα), and contains many ordered water molecules.


Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex.

Berke IC, Boehmer T, Blobel G, Schwartz TU - J. Cell Biol. (2004)

Structural alignment of the seven blades in the Nup133 β-propeller. (a and b) Superposition of 21 Cα positions from each blade calculated by the program MultiProt (http://bioinfo3d.cs.tau.ac.il/MultiProt/). Gray spheres mark the aligned Cα atoms from blade 3. Strand A lines the inner channel of the propeller and is roughly parallel to the pseudo-sevenfold axis. Blade 2 (orange), blade 5 (cyan), and blade 7 (purple) contain insertions. (c) Structure-based sequence alignment of Nup133 NTD blades. Conserved hydrophobic positions are shaded yellow. Clathrin NTD propeller blades are shown for comparison. The sequence is shown in Joy formatting (http://www-cryst.bioc.cam.ac.uk/~joy/). β-Strands are shown in blue, 310 helices are shown in maroon, capitalized residues are solvent inaccessible, bold indicates a sidechain-backbone amide H-bond, underline indicates sidechain-backbone carbonyl H-bond, and italics indicates a residue with positive phi values. Gray residues were not built in the model. Bracketed numbers refer to residues not depicted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172596&req=5

fig3: Structural alignment of the seven blades in the Nup133 β-propeller. (a and b) Superposition of 21 Cα positions from each blade calculated by the program MultiProt (http://bioinfo3d.cs.tau.ac.il/MultiProt/). Gray spheres mark the aligned Cα atoms from blade 3. Strand A lines the inner channel of the propeller and is roughly parallel to the pseudo-sevenfold axis. Blade 2 (orange), blade 5 (cyan), and blade 7 (purple) contain insertions. (c) Structure-based sequence alignment of Nup133 NTD blades. Conserved hydrophobic positions are shaded yellow. Clathrin NTD propeller blades are shown for comparison. The sequence is shown in Joy formatting (http://www-cryst.bioc.cam.ac.uk/~joy/). β-Strands are shown in blue, 310 helices are shown in maroon, capitalized residues are solvent inaccessible, bold indicates a sidechain-backbone amide H-bond, underline indicates sidechain-backbone carbonyl H-bond, and italics indicates a residue with positive phi values. Gray residues were not built in the model. Bracketed numbers refer to residues not depicted.
Mentions: We expressed the NTD of hNup133 (residues 67–514) in E. coli, crystallized it, and solved the structure to 2.35 Å. hNup133 NTD is a β-propeller with seven, four-stranded β-sheets arranged face to face around a central water-filled cavity (Fig. 2, a and b; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200408109/DC1). The polypeptide chain enters each propeller blade from the innermost strand and folds in an antiparallel manner (Fig. 2 a and Fig. 3 a, strands labeled A–D). Blade 7 of the propeller consists of the innermost three strands from the COOH terminus with the blade completed by the NH2 terminus of the domain. This 3 + 1 molecular clasp architecture is a common feature for stabilizing β-propellers (Paoli, 2001). The repeating antiparallel structure results in a top surface composed of the loops connecting strand D of one blade to strand A of the next (DA loop) as well as the BC loop within each blade, whereas the bottom surface is composed of the AB and CD loops. Two significant α-helical insertions are present in DA loops (Fig. 2, a and b, pink). The α1 helix inserts between the interface of blades 7 and 1, displacing blade 7 away and blade 1 toward the central axis. Blade 5 is extended and curls around helix α2 located in the DA loop connecting blades 4 and 5. This helical “wing” juts out from the core of the propeller by 15 Å. A disordered 20-residue insertion is present in the DA loop connecting blades 3 and 4 (DA34). The propeller has an overall diameter of 45–50 Å and the β-sheet core is ∼25-Å thick. Blade 2 has a short 310 helix just before strand 2A that projects into the top of the inner channel (Fig. 2 a; and Fig. 3 a, orange) and strand 2A is oriented away from the pseudo-sevenfold axis such that the bottom of the channel is wider than the top. The inner channel is oval shaped, being 12–16 Å wide at the top and 12–20 Å at the bottom (Cα to Cα), and contains many ordered water molecules.

Bottom Line: We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller.Other beta-propellers are predicted in a third of all nucleoporins.These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only approximately 30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller. The surface properties and conservation of the Nup133 beta-propeller suggest it may mediate multiple interactions with other proteins. Other beta-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

Show MeSH