Structural and functional analysis of an essential nucleoporin heterotrimer on the cytoplasmic face of the nuclear pore complex.

Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 09/2011; 108(40):16571-6. DOI: 10.1073/pnas.1112846108
Source: PubMed

ABSTRACT So far, only a few of the interactions between the ≈ 30 nucleoporins comprising the modular structure of the nuclear pore complex have been defined at atomic resolution. Here we report the crystal structure, at 2.6 Å resolution, of a heterotrimeric complex, composed of fragments of three cytoplasmically oriented nucleoporins of yeast: Nup82, Nup116, and Nup159. Our data show that the Nup82 fragment, representing more than the N-terminal half of the molecule, folds into an extensively decorated, seven-bladed β-propeller that forms the centerpiece of this heterotrimeric complex and anchors both a C-terminal fragment of Nup116 and the C-terminal tail of Nup159. Binding between Nup116 and Nup82 is mutually reinforced via two loops, one emanating from the Nup82 β-propeller and the other one from the β-sandwich fold of Nup116, each contacting binding pockets in their counterparts. The Nup82-Nup159 interaction occurs through an amphipathic α-helix of Nup159, which is cradled in a large hydrophobic groove that is generated from several large surface decorations of the Nup82 β-propeller. Although Nup159 and Nup116 fragments bind to the Nup82 β-propeller in close vicinity, there are no direct contacts between them, consistent with the noncooperative binding that was detected biochemically. Extensive mutagenesis delineated hot-spot residues for these interactions. We also showed that the Nup82 β-propeller binds to other yeast Nup116 family members, Nup145N, Nup100 and to the mammalian homolog, Nup98. Notably, each of the three nucleoporins contains additional nuclear pore complex binding sites, distinct from those that were defined here in the heterotrimeric Nup82•Nup159•Nup116 complex.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82-Nup159-Nsp1-Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery. © 2015 Gaik et al.
    The Journal of Cell Biology 02/2015; 208(3):283-97. DOI:10.1083/jcb.201411003 · 9.69 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A large family of G protein-coupled receptors (GPCRs) involved in cell adhesion has a characteristic autoproteolysis motif of HLT/S known as the GPCR proteolysis site (GPS). GPS is also shared by polycystic kidney disease proteins and it precedes the first transmembrane segment in both families. Recent structural studies have elucidated the GPS to be part of a larger domain named GPCR autoproteolysis inducing (GAIN) domain. Here we demonstrate the remote homology relationships of GAIN domain to ZU5 domain and Nucleoporin98 (Nup98) C-terminal domain by structural and sequence analysis. Sequence homology searches were performed to extend ZU5-like domains to bacteria and archaea, as well as new eukaryotic families. We found that the consecutive ZU5-UPA-death domain domain organization is commonly used in human cytoplasmic proteins with ZU5 domains, including CARD8 (caspase recruitment domain-containing protein 8) and NLRP1 (NACHT, LRR and PYD domain-containing protein 1) from the FIIND (Function to Find) family. Another divergent family of extracellular ZU5-like domains was identified in cartilage intermediate layer proteins and FAM171 proteins. Current diverse families of GAIN domain subdomain B, ZU5 and Nup98 C-terminal domain likely evolved from an ancient autoproteolytic domain with an HFS motif. The autoproteolytic site was kept intact in Nup98, p53-induced protein with a death domain and UNC5C-like, deteriorated in many ZU5 domains and changed in GAIN and FIIND. Deletion of the strand after the cleavage site was observed in zonula occluden-1 and some Nup98 homologs. These findings link several autoproteolytic domains, extend our understanding of GAIN domain origination in adhesion GPCRs and provide insights into the evolution of an ancient autoproteolytic domain. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Journal of Molecular Biology 10/2014; 426(24). DOI:10.1016/j.jmb.2014.10.011 · 3.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We introduce toleranced models (TOMs), a generic and versatile framework meant to handle models of macromolecular assemblies featuring uncertainties on the shapes and the positions of proteins. A TOM being a continuum of nested shapes, the inner (resp. outer) ones representing high (low) confidence regions, we present topological and geometric statistics assessing features of this continuum at multiple scales. While the topological statistics qualify contacts between instances of protein types and complexes involving prescribed protein types, the geometric statistics scale the geometric accuracy of these complexes. We validate the TOM framework on recent average models of the entire nuclear pore complex (NPC) obtained from reconstruction by data integration, and confront our quantitative analysis against experimental findings related to complexes of the NPC, namely the Y-complex, the T-complex, and the Nsp1-Nup82-Nup159 complex. In the three cases, our analysis bridges the gap between global qualitative models of the entire NPC, and atomic resolution models or putative models of the aforementioned complexes. In a broader perspective, the quantitative assessments provided by the TOM framework should prove instrumental to implement a virtuous loop "model reconstruction-model selection", in the context of reconstruction by data integration.
    Proteins Structure Function and Bioinformatics 08/2012; 80(9):2125-36. DOI:10.1002/prot.24092 · 2.92 Impact Factor