The symmetric core of the nuclear pore complex can be considered schematically as a series of concentric cylinders. A peripheral cylinder coating the pore membrane contains the previously characterized, elongated heptamer that harbors Sec13-Nup145C in its middle section. Strikingly, Sec13-Nup145C crystallizes as a hetero-octamer in two space groups. Oligomerization of Sec13-Nup145C was confirmed biochemically. Importantly, the numerous interacting surfaces in the hetero-octamer are evolutionarily highly conserved, further underlining the physiological relevance of the oligomerization. The hetero-octamer forms a slightly curved, yet rigid rod of sufficient length to span the entire height of the proposed membrane-adjacent cylinder. In concordance with the dimensions and symmetry of the nuclear pore complex core, we suggest that the cylinder is constructed of four antiparallel rings, each ring being composed of eight heptamers arranged in a head-to-tail fashion. Our model proposes that the hetero-octamer would vertically traverse and connect the four stacked rings.

We recently proposed a cylindrical coat for the nuclear pore membrane in the nuclear pore complex (NPC). This scaffold is generated by multiple copies of seven nucleoporins. Here, we report three crystal structures of the nucleoporin pair Seh1*Nup85, which is part of the coat cylinder. The Seh1*Nup85 assembly bears resemblance in its shape and dimensions to that of another nucleoporin pair, Sec13*Nup145C. Furthermore, the Seh1*Nup85 structures reveal a hinge motion that may facilitate conformational changes in the NPC during import of integral membrane proteins and/or during nucleocytoplasmic transport. We propose that Seh1*Nup85 and Sec13*Nup145C form 16 alternating, vertical rods that are horizontally linked by the three remaining nucleoporins of the coat cylinder. Shared architectural and mechanistic principles with the COPII coat indicate a common evolutionary origin and support the notion that the NPC coat represents another class of membrane coats.

Summary Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins’ function is poorly understood. Here, we combine optical-trapping with TIRF-based microscopy to measure the force-dependence of microtubule interaction for three non-motor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase non-linearly with MAP velocity across microtubules and depend on filament polarity, with NuMA’s friction being lower when moving towards minus-ends, EB1’s lower towards plus-ends, and PRC1 exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within active microtubule pairs they crosslink. Our findings reveal how non-motor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures.

SUMMARY Microtubule organization depends on the γ-Tubulin ring complex (γ-TuRC), a ~2.3-MDa nucleation factor comprising an asymmetric assembly of γ-Tubulin and GCP2-GCP6. However, it is currently unclear how the γ-TuRC-associated microproteins MZT1 and MZT2 contribute to the structure and regulation of the holocomplex. Here, we report cryo-EM structures of MZT1 and MZT2 in the context of the native human γ-TuRC. MZT1 forms two subcomplexes with the N-terminal α-helical domains of GCP3 or GCP6 (GCP-NHDs) within the γ-TuRC “lumenal bridge.” We determine the X-ray structure of recombinant MZT1/GCP6-NHD and find it is similar to that within the native γ-TuRC. We identify two additional MZT/GCP-NHD-like subcomplexes, one of which is located on the outer face of the γ-TuRC and comprises MZT2 and GCP2-NHD in complex with a centrosomin motif 1 (CM1)-containing peptide. Our data reveal how MZT1 and MZT2 establish multi-faceted, structurally mimetic “modules” that can expand structural and regulatory interfaces in the γ-TuRC.

Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, via its hDgt6 subunit's C-terminus, to nucleate microtubules within spindles. However, augmin's biochemical complexity has restricted analysis of its structural organization and function. Here, we reconstitute human augmin and show it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains in vitro microtubule-binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to Xenopus egg extracts promotes microtubule aster formation, an activity enhanced by Ran-GTP. This activity requires microtubule binding, but not the characterized hDgt6 γ-tubulinrecruitment domain. Tetrameric sub-complexes induce asters, but activity and microtubule bundling within asters are reduced compared to octameric complexes. Together, our findings shed light on augmin's structural organization, microtubule binding properties and define subunits required for its function in organizing microtubule-based structures.

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The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a bba-metal topology. Here we report the crystal structures of a Zn . ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg 2+ at concentrations of <25 mM, indicating that zinc is the physiologically relevant metal ion in N-ColE7 in host E. coli. In the crystal structure of N-ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni 2+ is bound in N-ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His-metal finger motif can be coordinated to six ligands, such as Mg 2+ in I-PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni 2+ or Zn 2+ in ColE7. Universally, the metal ion in the His-metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P-O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.

The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclear pore complex. Here, we present the crystal structure of the heterotrimeric Sec13⅐Nup145C⅐Nup84 complex, the centerpiece of the heptamer, at 3.2-Å resolution. Nup84 forms a U-shaped ␣-helical solenoid domain, topologically similar to two other members of the heptamer, Nup145C and Nup85. The interaction between Nup84 and Nup145C is mediated via a hydrophobic interface located in the kink regions of the two solenoids that is reinforced by additional interactions of two long Nup84 loops. The Nup84 binding site partially overlaps with the homo-dimerization interface of Nup145C, suggesting competing binding events. Fitting of the elongated Z-shaped heterotrimer into electron microscopy (EM) envelopes of the heptamer indicates that structural changes occur at the Nup145C⅐Nup84 interface. Docking the crystal structures of all heptamer components into the EM envelope constitutes a major advance toward the completion of the structural characterization of the Nup84 complex.electron microscopy docking ͉ nuclear pore complex ͉ protein-protein interaction ͉ X-ray crystallography ͉ binding promiscuity