Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex

Abstract

Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical crosslinking with mass spectrometric readout. In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex, a stable hetero-heptameric assembly (ϳ600 kDa), 16 copies of which form the outer rings of the 50-MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope. Molecular & Cellular Proteomics

Integrative structure and functional anatomy of a nuclear pore complex

“…2–3C). To inform the proximities, orientations, and conformations of the Nups, isolated NPCs were subjected to cross-linking with MS readout 9,10 . This approach identified 3,077 unique cross-linked pairs of residues, providing distance restraints between them, both within and between Nups (Fig.…”

“…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).…”

“…After reduction and alkylation, the cross-linked complexes were separated by 3–8% SDS-PAGE (NuPAGE Tris-Acetate Fisher) to reduce the complexity of the sample. For in-gel digestion, the high molecular weight region gel bands (> 460 kDa, estimated by the high MW protein markers, Invitrogen) corresponding to the cross-linked NPC proteins were sliced and proteolysed by trypsin as previously described 66,67 . Briefly, gel plugs were crushed into small pieces, ~5–10 µg of sequencing grade trypsin (Promega) per ~100 µg protein was added with subsequent 6 – 8 hour incubation.…”

“…The initial search results were obtained using a default 5% false discovery rate (FDR) expected by target-decoy search strategy. All spectra were manually verified as previously described 66,67,70–72 . The cross-linking data was analyzed and plotted by an on-line software tool of CX-Circos (http://cx-circos.net; manuscript in preparation) (Fig.…”