Figure 1. Assembly of a Nup82NTD•Nup159T•Nup116CTD heterotrimer. (A) Domain organization of yeast Nup82, Nup116, and Nup159 (1). Bars denote the fragments that were used for heterotrimer assembly and crystallization: Nup82 N-terminal domain (NTD, blue); Nup116 C-terminal domain (CTD, green); and Nup159 C-terminal tail (T, red). GLEBS, binding site for the RNA export factor Gle2; DID, dynein light chain interacting domain. (B) Analysis by size exclusion chromatography coupled to multiangle light scattering. The differential refractive indices of Nup116CTD (red), Nup82NTD•Nup159T (blue), and Nup82NTD•Nup159T•Nup116CTD (green) are plotted against the elution volumes from a Superdex 200 10/300 GL gel filtration column (GE Healthcare) with dots indicating molar masses.

Yoshida, K., Seo, H.S., Debler, E.W., Blobel, G., Hoelz, A.

(2011). Proc. Natl. Acad. Sci. USA, 108, 16571-16576.

Figure 2. Structural overview of the Nup82NTD•Nup159T•Nup116CTD complex. (A–C) Ribbon representation from various angles. Nup82NTD (blue) folds into a β-propeller with various noncanonical insertions highlighted in yellow (3D4A or “FGL” loop), gray (4CD), and orange (6CD); Nup116CTD (green) is a β-sandwich with a β6-αB connector (“K-loop”) indicated in magenta; Nup159T (red) folds into an amphipathic α-helix. Dotted lines represent disordered regions. (D) Schematic representation of the β-propeller of Nup82NTD. Prominent insertions and secondary structure elements are labeled. The asterisk denotes the N-terminal region that fits into the crevice between blades 1 and 7, replacing the canonical Velcro closure observed in most β-propellers.

Figure 3. Close-up view of the Nup82-Nup116 and Nup82-Nup159 interfaces. Nup82NTD interacts with Nup116CTD via two adjacent sites. (A) One site is formed by the 3D4A “FGL loop” of Nup82NTD, which binds to a groove of Nup116CTD between helix αB and strand β5. (B) The other site is formed by the “K-loop” of Nup116CTD, K1063 of which binds to a Nup82 pocket with an aspartate (D204) at its bottom and a hydrophobic bracelet at its entry. (C) Nup159T binds to a groove in Nup82NTD that is formed by the 4CD and 6CD insertions and blade 5. Color code as in Fig. 2.

Figure 4. Human Nup98CTD binds to yeast Nup82NTD. (A) Domain organization of human Nup98 (1, 18) indicating the C-terminal domain (CTD, green) and the auto-proteolytic 6-kDa protein (gray), as well as the auto-proteolytic cleavage site (red arrow). (B) Size exclusion chromatography analysis of purified yeast Nup82NTD•Nup159T (blue), human Nup98CTD•6-kDa (red) and a mixture (green) in an ≈1∶2 ratio. Note formation of a trimeric Nup82NTD•Nup159T•hNup98CTD complex leads to displacement of the 6-kDa protein (inset). (C) Fractions, indicated by gray bars in (B), of hNup98CTD•6-kDa (top), Nup82NTD•Nup159T (center), and their mixture (bottom) were analyzed by SDS-PAGE; proteins are indicated on the right and molecular weight standards on the left.

Figure 5. Binding of yeast paralogs of Nup116CTD to Nup82NTD. Interaction analysis between Nup82NTD•Nup159T and the yeast Nup116CTD homologs (A) Nup100CTD and (B) Nup145NCTD. Size exclusion chromatography analysis of Nup82NTD•Nup159T (blue), Nup100CTD (red) and Nup145NCTD (red), and their mixtures (green).

Figure 6. In vivo analysis of Nup82 mutants. (A) Domain organization of the GFP-labeled Nup82 constructs, colored as in Fig. 1A. The black and red arrows indicate the positions of the DFY and LILLF mutations, respectively. (B) Yeast growth analysis using a nup82Δ strain transformed with the indicated GFP-Nup82 constructs. 10-fold serial dilutions were spotted on SD-Leu plates and grown for 2–3 d at the indicated temperatures. The combination of the DFY and LILLF mutations in one mutant (Nup82DFY–LILLF) has the same effect on growth as the deletion of the entire NTD (Nup82ΔNTD). (C) In vivo localization of the GFP-Nup82 constructs at 37 °C. Both the Nup82DFY–LILLF and the Nup82ΔNTD mutants fail to localize to the nuclear rim. The scale bars represent 5 μm.

Figure S1.Structural comparison of Nup116CTD •Nup82NTD and hNup98CTD •6-kDa heterodimers. (A) Ribbon representation of Nup116CTD (green) in complex with Nup82NTD (yellow). For clarity, only the Nup82NTD 3D4A (or “FGL”) loop is shown. The Nup116CTD β6-αB connector (“K-loop”) that interacts with Nup82NTD is colored in magenta. (B) Ribbon representation of hNup98CTD (gray), the human homolog of yeast Nup116, in complex with an N-terminal segment of the auto- proteolytic 6-kDa fragment (orange). (C) Superposition of the Nup116CTD•Nup82NTD and hNup98CTD•6-kDa heterodimers illustrates that the Nup82 FGL loop binds to Nup116 in the same fashion as the 6-kDa fragment to hNup98, providing a molecular basis for the mutually exclusive binding of Nup82 and 6-kDa fragment to Nup116 (Fig. 4). (D) Detailed view of the interactions of Nup116CTD with the Nup82NTD FGL loop (yellow) and the 6-kDa YGL loop (orange). For clarity, hNup98CTD superimposed on Nup116 (green) is omitted.

Figure S2.Surface properties of Nup116CTD . (A) Surface rendition of Nup116CTD . The two binding sites for Nup82NTD are colored in yellow and blue, respectively. The position of the invariant K1063 in the K-loop is indicated. (B) Sequence conservation of Nup116CTD based on the alignment in Fig. S5, mapped onto the surface and colored in a gradient from white (less than 40% similarity) to yellow (40% similarity) to red (100% identity). (C) Surface rendition of Nup116CTD, colored according to the electrostatic potential, ranging from red (−10 kBT∕e) to blue (þ10 kBT∕e). While the site with the projecting K-loop is positively charged, the other site, which accommodates the FGL loop, is primarily hydrophobic.

Figure S3.Surface properties of Nup82NTD. (A) Surface rendition of Nup82NTD. The Nup116CTD and Nup159T binding sites are colored in green and red, respec- tively. The positions of D204, F290, and Y295, which disrupt Nup116CTD binding, and of L393, I397, L402, L405, and F410, which interfere with the Nup159T interaction, are indicated in yellow and orange, respectively. As a reference, the ribbon representation of the heterotrimer is shown on the left. (B) Sequence conservation of Nup82NTD based on the alignment in Fig. S4, mapped onto the surface and colored in a gradient from white (less than 40% similarity) to yellow (40% similarity) to red (100% identity). (C) Surface rendition of Nup82NTD, colored according to the electrostatic potential, from red (−10 kBT∕e) to blue (þ10 kBT∕e). The Nup116 binding site is strongly negatively charged, consistent with the key aspartate residue (D204).

Figure S4.Multispecies sequence alignment for the NTD of Nup82 homologs. The numbering below the alignment is relative to S. cerevisiae Nup82. The overall sequence conservation at each position is shaded in a color gradient from white (less than 40% similarity) to dark red (100% identity) using the Blosum62 weighting algorithm. The secondary structure is indicated above the sequence as blue boxes (α-helices), green arrows (β-strands), gray lines (coil regions), and gray dots (disordered residues). Residues involved in the interaction with Nup116CTD and Nup159T are indicated by green and magenta dots, respectively. The positions of D204, F290, and Y295 and of L393, I397, L402, L405, and F410 are labeled below the dots.

Figure S5.Multispecies sequence alignment for the CTD of Nup116 homologs. The numbering below the alignment is relative to S. cerevisiae Nup116. The overall sequence conservation at each position is shaded in a color gradient from white (less than 40% similarity) to dark red (100% identity) using the Blosum62 weighting algorithm. The secondary structure is indicated above the sequence as blue boxes (α-helices), green arrows (β-strands), and gray dots (disordered residues). The positions of the invariant K1063 and of A1110 which corresponds to the catalytic S864 in hNup98 are indicated by an asterisk and an arrowhead, respectively. The two bottom sequences refer to the Nup116 homologs in S. cerevisiae, Nup100 and Nup145N. Consistent with the auto-proteolytic biogenesis of Nup145N, A1110 of Nup116 is replaced by a catalytic serine residue.

Figure S6.Isothermal titration calorimetry (ITC) analysis. (A–C) ITC measurements of Nup82NTD-mediated interactions. The upper parts of the boxes show raw data, while the lower parts refer to the integrated heat changes, corrected for the heat of dilution, and the fitted curve based on a single-site model. The first component of the reaction pair is titrated into the second component. (D) Summary of the ITC results. The dissociation constant (Kd ), binding enthalpy (ΔH), entropy (ΔS), and the stoichiometry (N) were derived by curve fitting using a single-site model. Note that deletion of the 3D4A (“FGL”) loop in Nup82NTD only leads to a fivefold reduction in affinity for Nup116CTD with respect to wild type and that the presence or absence of Nup159T minimally affects the Nup82NTD-Nup116CTD interaction.

Figure S7.In vivo analysis of Nup116 mutants. (A) Domain organizations of the GFP-labeled Nup116 constructs, colored as in Fig. 1A. (B) Yeast growth analysis using a nup116Δ strain transformed with the indicated GFP-Nup116 constructs. 10-fold serial dilutions were spotted on SD-Leu plates and grown for 2–3 d at the indicated temperatures. (C) In vivo localization of GFP-Nup116 at 37 °C. Consistent with the finding that removal of the crystallized fragment, Nup116ΔCTD , does not abolish nuclear rim staining, an unstructured region upstream is identified as another nuclear envelope targeting region. The scale bars represent 5 μm.

PDB coordinates (link to PDB site)

PDB coordinates (.pdb)

Structure factors (.txt)

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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.

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

Figure S8.In vivo analysis of Nup159 mutants. (A) Domain organizations of GFP-labeled Nup159 constructs, colored as in Fig. 1A. (B) Yeast growth analysis using a nup159Δ strain transformed with the indicated GFP-Nup159 constructs. 10-fold serial dilutions were spotted on SD-Leu plates and grown for 2–3 d at the indicated temperatures. (C) In vivo localization of GFP-Nup159 at 37 °C. A mutant lacking the crystallized tail fragment appears identical to wild type, while a larger C-terminal truncation leads to lethality at 37 °C and mistargeting. The scale bars represent 5 μm.