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Technology Topics Hybrid/Integrative Methods

Nuclear Pore Complex: Integrative Approach to Probe Nup133

SBKB [doi:10.1038/sbkb.2014.242]
Featured Article - February 2015
Short description: The integration of three structural methods offers insight into membrane association of the NPC.

Integrative modeling of ScNup133 reveals the population weight averages of four states, a major extended conformation and three minor compact conformations. Figure from ref. 1 , © 2014 The American Society for Biochemistry and Molecular Biology.

Linking the nuclear and cytoplasmic compartments, the nuclear pore complex (NPC) is one of the cell's most essential and complex structures. Using an approach that integrates X-ray crystallography, small angle X-ray scattering (SAXS) and electron microscopy (EM) data, Almo, Rout, Sali and colleagues have produced atomic-level models for Saccharomyces cerevisiae (Sc) Nup133, a major component of the Nup84 complex in the NPC's outer ring. This work, from two PSI Centers (PSI NYSGRC and NPCXstals), aimed to solve the structure of this notoriously flexible protein and determine whether Nup133 contains a motif postulated to interact with the nuclear membrane.

Aided by the crystal structure of a related fungal VpNup13355–502 (PDB 4Q9T) and SAXS data, the authors built an initial model for full-length ScNup133. However, as the SAXS profile obtained for ScNup1332–1157 did not agree with that arising from the comparative model, the authors looked more carefully at the solution dynamics of ScNup1332–1157 and concluded that the data could only be explained by a multistate model. Comparison of the SAXS data and EM class averages resulted in a final model with four states, distinguished mainly by the relative positions of the N- and C-terminal domains; these states were confirmed by crosslinking/mass spectrometry and mutational analyses.

One feature noted in previous Nup133 structures was an unresolved loop in the seven-bladed β-propeller; it had been speculated to be an ArfGAP1 lipid packing sensor (ALPS) motif, which senses and stabilizes membrane curvature. Using a helical wheel representation, the ALPS motif was identified in ScNup133, as well as two copies in ScNup120, an ortholog that is also part of the Nup84 complex. The integrative approach outlined in this study permitted the authors to determine the structure of the major open state as well as three minor compact forms. In addition, the ability to isolate the ALPS motif in both Nup133 and Nup120 establishes how the Nup84 complex may be anchored within the nuclear envelope membrane, and rebuts the previous suggestion that only organisms with open mitosis may require such membrane contacts. These results also classify Nup133 and Nup120 as coat-like proteins, with the ALPS being an ancestral remnant of vesicle-coating complexes.

Angela K. Eggleston

References

  1. S.J. Kim et al. Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex.
    Mol Cell Proteomics 13, 2911-26 (2014). doi:10.1074/mcp.M114.040915

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