Integrative Structure–Function Mapping of the Nucleoporin Nup133 Suggests a Conserved Mechanism for Membrane Anchoring of the Nuclear Pore Complex

Abstract

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133 55-502 ) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup133 2-1157 . Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes. Molecular & Cellular Proteomics 13:

Integrative structure and functional anatomy of a nuclear pore complex

“…3A–B, 6A). Individual Nups, their domains, and subcomplexes were represented based on published crystallographic structures, integrative structures, and comparative models 9,10,12 (Supplementary Table 2; Methods), validated by SAXS profiles for 18 Nups (147 constructs; Supplementary Table 6). An ensemble of structural solutions for the NPC that sufficiently satisfied all experimental data was calculated by extensive configurational sampling 8,9 (Supplementary Table 3; Methods).…”

“…The outer rings also help define the overall height of the NPC, such that it is appropriate for the width of the NE. Each Nup84 complex is anchored to the pore membrane by MBMs situated within the N-terminal β-propellers of Nup133 and Nup120 10,12,21 (Fig. 5D).…”

“…SAXS data were collected at concentrations ranging from 0.5 to 5.0 (or higher, depending on the sample) mg/mL, using the previously defined standard protocol 43,88,90 ; ~20 one-second exposures were used for each sample and buffers maintained at 15°C. Further details of the SAXS experiments are provided in our previous publications 43,88,90 .…”

“…The integrative structure modeling protocol ( i.e ., stages 2, 3, and 4) was scripted using the Python Modeling Interface (PMI) package, version 4d97507, a library for modeling macromolecular complexes based on our open-source Integrative Modeling Platform (IMP) package 98 , version 2.6 (https://integrativemodeling.org). The current procedure is an updated version of previously described protocols 66,72,90,101–104 . Files containing the input data, scripts, and output results are available at https://salilab.org/npc2018.…”

“…Re-interpreted immuno-EM data 48,97 informed the positions of 29 Nups. Predictions of the transmembrane domains obtained from SGD 105 ( Saccharomyces Genome Database; http://yeastgenome.org) and predictions of membrane binding motifs by the HeliQuest webserver 90,106 informed about their proximity to the pore membrane. Previous immuno-EM measurements 107 informed the end-to-end distance for Mlp1 and Mlp2.…”