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Protein Interaction Networks: When the Sum Is Greater than the Parts

SBKB [doi:10.1038/sbkb.2012.133]
Featured Article - April 2013
Short description: Integration of low-resolution approaches yields functional and evolutionary insights into the NPC.

The Nup84 complex. (a) Localization density map, with two ribbon structures from ensemble fitted. (b) Improving our understanding of Nup84: colored areas indicate comparative model use; the arm with components now correctly positioned is traced, and potential Seh1-Sec13 interaction surface is shown. Figure courtesy of Javier Fernandez-Martinez.

The yeast nuclear envelope contains roughly 150 nuclear pore complexes (NPCs) which allow the traffic of molecules between the nucleus and cytoplasm. NPCs are highly organized 40–60 MDa structures containing multiple copies of ∼30 proteins called nucleoporins (nups). The NPC is comprised of several coaxial rings, and the outermost of these rings contains the Y-shaped, seven-member Nup84 complex: Nup133, Nup84, Nup145c and Sec13 form its base, and Nup85, Seh1 and Nup120 comprise its arms. Although atomic resolution structures for some parts of the Nup84 complex exist, they do not offer a full description of the subunits or domain interfaces that form the complex or of the complex's orientation within the NPC.

Realizing that such questions could not be answered using a single approach, the laboratories of Michael Rout, Andrej Sali, Brian Chait and David Stokes (collaborators in the Nucleocytoplasmic transport PSI biology partnership with the NYSGRC ) computationally integrated a diverse set of experimental data from a number of different sources, with resolutions spanning several orders of magnitude. Domains of the proteins in the Nup84 complex were systematically deleted to define their connectivity and relative orientation by immunopurification and mass spectrometry analyses. The general shape of the complex and the position of certain components were determined by negative-stain electron microscopy. Available crystal structures or comparative models were used to represent each subunit. Experimental data were translated into spatial restraints, which were then used to calculate an ensemble of solutions. Structures that met all the restraints underwent rounds of additional refinement and optimization. The almost 9,500 structures that emerged from this process clustered into a single ensemble of solutions, with a defined precision of 1.5nm.

The overall structure of the ensemble model is not vastly different than those previously suggested, but several new aspects of the Nup84 complex are revealed. The longer arm of the Y contains only Nup120, connected to the rest of the complex by its C-terminal region in a “hub.” The shorter arm contains Seh1 and Nup85, whose C terminus is part of the hub; this dimer faces outward, an orientation different from that based on some prior analyses, but consistent with other existing data. The lower part of the hub is formed by the C-terminus of Nup145c and by its N-terminus, which was previously predicted to be disordered. The remainder of Nup145c initiates the stalk and connects to Nup84. At the other end, Nup84 connects tail-to-tail to Nup133.

In terms of overall position of the Nup84 complex within the NPC, the ensemble model indicates that the eight complexes within the outer ring are arranged in a head-to-tail fashion. Analysis of the effects of domain deletions on the stability of Nup84 complex interaction with the ring suggests that the complex makes multiple, cooperative weak contacts, primarily via its two arms.

The truncation mutants were also used to generate phenotypic profiles that were quantified and mapped onto the structure. The phenotypes analyzed were fitness (growth) and NPC clustering. The greatest effects on fitness were seen in mutants affecting the tips of the arms and the middle of the stalk, where it is suggested that they form connections to the NPC core. In contrast, the effects on NPC clustering were limited to the Nup120 and Nup133 truncations, and it is proposed that these regions are involved in stabilizing the curvature of the nuclear pore membrane.

Finally, the connections between the subunits lend support for the protocoatomer hypothesis, which proposes a single common ancestor for all coatomer-like complexes, including the Nup84 complex. However, whereas other vesicle-coating and tethering complexes form symmetric homooligomers, the Nup84 complex, via a process of gene duplication, diversification and likely secondary loss, is asymmetric and heterooligomeric. The authors speculate that these features allow the Nup84 complex to tune the membrane structure to form the pore, rather than forming a cage.

Angela K. Eggleston

References

  1. J. Fernandez-Martinez et al. Structure-function mapping of a heptameric module in the nuclear pore complex.
    J. Cell Biol. 196, 419-434 (2012). doi:10.1083/jcb.201109008

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