Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex - PubMed (original) (raw)

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

Ian C Berke et al. J Cell Biol. 2004.

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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Figures

Figure 1.

Figure 1.

Domain structure of Nup133. (a) Two domains were predicted, with the NTD having helical and sheet content, and the CTD being all helical. The construct used for crystallization is boxed. (b) In vitro binding of hNup133 domains to recombinant GST-hNup107. Top and bottom panels show the bound and unbound fractions of various [35S]methionine-labeled Nup133 translation products incubated with recombinant GST-Nup107 immobilized on affinity resin. (c) Localization of EGFP-tagged hNup133 domains in HeLa cells. The CTD shows punctate rim staining that overlaps with the mAb414 signal. The NTD shows diffuse EGFP-signal, with a weak concentration at the nuclear rim.

Figure 2.

Figure 2.

Architecture of hNup133 NTD. (a) Ribbon representation of hNup133 NTD β-propeller, top view. The four strands of each blade are labeled A–D. Blades are labeled 1–7 and colored red through purple with helical insertions shown in pink. The disordered loops are represented as transparent tubes. (b) Side views of the propeller related ∼120° to each other. Figures were prepared with Dino (

http://www.dino3d.org

) and Povray (

http://www.povray.org

).

Figure 3.

Figure 3.

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.

Figure 4.

Figure 4.

Conserved features of the Nup133 NTD propeller. (a) Surface representation showing conservation of residues on the propeller sides. Each view is an ∼120° rotation about the pseudo-sevenfold axis beginning from the NH2 and COOH termini in blade 7 and is the same as in Fig. 2 b. The tube marks the region of the disordered DA34 loop. Conservation scores were calculated using the ConSurf Server (

http://consurf.tau.ac.il/

) and colored maroon (most conserved) to cyan (most variable). (b) Electrostatic potential of the Nup133 β-propeller. Orientation is as in part a. Red indicates negative regions of the potential and blue indicates positive regions. (c) Sequence alignment of the DA34 loop across representative metazoans. Position of the conserved protein kinase A consensus site is boxed and potential phosphorylated residues are marked by yellow stars. Green triangles mark conserved hydrophobic positions.

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