André Hoelz

The Structure of the Nuclear Pore Complex

In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies.

Structural Evidence for Feedback Activation by Ras·GTP of the Ras-Specific Nucleotide Exchange Factor SOS

Growth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras.GTP. The crystal structures suggest that Ras.GTP stabilizes the active site of SOS allosterically, and we show that Ras.GTP forms ternary complexes with SOS(cat) in solution and increases significantly the rate of SOS(cat)-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras.

Crystal Structure of a Tetradecameric Assembly of the Association Domain of Ca2+/Calmodulin-Dependent Kinase II

We report the crystal structure of the 143 residue association domain of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The association domain forms a hub-like assembly, composed of two rings of seven protomers each, which are stacked head to head and held together by extensive interfaces. The tetradecameric organization of the assembly was confirmed by analytical ultracentrifugation and multiangle light scattering. Individual protomers form wedge-shaped structures from which N-terminal helical segments that connect to the kinase domain extend toward the equatorial plane of the assembly, consistent with the arrangement of the kinase domains in a second outer ring. A deep and highly conserved pocket present within the association domain may serve as a docking site for proteins that interact with CaMKII.

Architecture of a coat for the nuclear pore membrane.

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.

A Fence-like Coat for the Nuclear Pore Membrane

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.

Structure of Nup58/45 suggests flexible nuclear pore diameter by intermolecular sliding.

The nucleoporins Nup58 and Nup45 are part of the central transport channel of the nuclear pore complex, which is thought to have a flexible diameter. In the crystal structure of an α-helical region of mammalian Nup58/45, we identified distinct tetramers, each consisting of two antiparallel hairpin dimers. The intradimeric interface is hydrophobic, whereas dimer-dimer association occurs through large hydrophilic residues. These residues are laterally displaced in various tetramer conformations, which suggests an intermolecular sliding by 11 angstroms. We propose that circumferential sliding plays a role in adjusting the diameter of the central transport channel.

Oligomerization states of the association domain and the holoenyzme of Ca2+/CaM kinase II

Ca2+/calmodulin activated protein kinase II (CaMKII) is an oligomeric protein kinase with a unique holoenyzme architecture. The subunits of CaMKII are bound together into the holoenzyme by the association domain, a C‐terminal region of ≈ 140 residues in the CaMKII polypeptide. Single particle analyses of electron micrographs have suggested previously that the holoenyzme forms a dodecamer that contains two stacked 6‐fold symmetric rings. In contrast, a recent crystal structure of the isolated association domain of mouse CaMKIIα has revealed a tetradecameric assembly with two stacked 7‐fold symmetric rings. In this study, we have determined the crystal structure of the Caenorhabditis elegans CaMKII association domain and it too forms a tetradecamer. We also show by electron microscopy that in its fully assembled form the CaMKII holoenzyme is a dodecamer but without the kinase domains, either from expression of the isolated association domain in bacteria or following their removal by proteolysis, the association domains form a tetradecamer. We speculate that the holoenzyme is held in its 6‐fold symmetric state by the interactions of the N‐terminal ≈ 1–335 residues and that the removal of this region allows the association domain to convert into a more stable 7‐fold symmetric form.

Structural and functional analysis of the interaction between the nucleoporin Nup214 and the DEAD-box helicase Ddx19.

Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADP-bound state at 2.5 Å resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export.

Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex

The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we present the crystal structure of one of its 7 components, Nup120, which reveals a β propeller and an α-helical domain representing a novel fold. We discovered a previously unidentified interaction of Nup120 with Nup133 and confirmed the physiological relevance in vivo. As mapping of the individual components in the Nup84 complex places Nup120 and Nup133 at opposite ends of the heptamer, our findings indicate a head-to-tail arrangement of elongated Nup84 complexes into a ring structure, consistent with a fence-like coat for the nuclear pore membrane. The attachment site for Nup133 lies at the very end of an extended unstructured region, which allows for flexibility in the diameter of the Nup84 complex ring. These results illuminate important roles of terminal unstructured segments in nucleoporins for the architecture, function, and assembly of the NPC.

Architecture of the symmetric core of the nuclear pore

Blueprint for a macromolecular machine Nuclear pore complexes (NPCs) consist of around 1000 protein subunits, are embedded in the membrane that surrounds the nucleus, and regulate transport between the nucleus and the cytoplasm. Although the overall shape of NPCs is known, the details of this macromolecular complex have been obscure. Now, Lin et al. have reconstituted the pore components, determined the interactions between them, and fitted them into a tomographic reconstruction. Kosinski et al. have provided an architectural map of the inner ring of the pore. Science, this issue pp. 10.1126/science.aaf1015 and 363 Reconstitution, spectroscopy, and crystallography allow the construction of a model of the human nuclear pore. INTRODUCTION The nuclear pore complex (NPC) is the primary gateway for the transport of macromolecules between the nucleus and cytoplasm, serving as both a critical mediator and regulator of gene expression. NPCs are very large (~120 MDa) macromolecular machines embedded in the nuclear envelope, each containing ~1000 protein subunits, termed nucleoporins. Despite substantial progress in visualizing the overall shape of the NPC by means of cryoelectron tomography (cryo-ET) and in determining atomic-resolution crystal structures of nucleoporins, the molecular architecture of the assembled NPC has thus far remained poorly understood, hindering the design of mechanistic studies that could investigate its many roles in cell biology. RATIONALE Existing cryo-ET reconstructions of the NPC are too low in resolution to allow for de novo structure determination of the NPC or unbiased docking of nucleoporin fragment crystal structures. We sought to bridge this resolution gap by first defining the interaction network of the NPC, focusing on the evolutionarily conserved symmetric core. We developed protocols to reconstitute NPC protomers from purified recombinant proteins, which enabled the generation of a high-resolution biochemical interaction map of the NPC symmetric core. We next determined high-resolution crystal structures of key nucleoporin interactions, providing spatial restraints for their relative orientation. By superposing crystal structures that overlapped in sequence, we generated accurate full-length structures of the large scaffold nucleoporins. Lastly, we used sequential unbiased searches, supported by the biochemical data, to place the nucleoporin crystal structures into a previously determined cryo-ET reconstruction of the intact human NPC, thus generating a composite structure of the entire NPC symmetric core. RESULTS Our analysis revealed that the inner and outer rings of the NPC use disparate mechanisms of interaction. Whereas the structured coat nucleoporins of the outer ring form extensive surface contacts, the scaffold proteins of the inner ring are bridged by flexible sequences in linker nucleoporins. Our composite structure revealed a defined spoke architecture in which each of the eight spokes spans the nuclear envelope, with limited cross-spoke interactions. Most nucleoporins are present in 32 copies, with the exceptions of Nup170 and Nup188, which are present in 48 and 16 copies, respectively. Lastly, we observed the arrangement of the channel nucleoporins, which orient their N termini into two 16-membered rings, thus ensuring that their N-terminal FG repeats project evenly into the central transport channel. CONCLUSION Our composite structure of the NPC symmetric core can be used as a platform for the rational design of experiments to investigate NPC structure and function. Each nucleoporin occupies multiple distinct biochemical environments, explaining how such a large macromolecular complex can be assembled from a relatively small number of genes. Our integrated, bottom-up approach provides a paradigm for the biochemical and structural characterization of similarly large biological mega-assemblies. Composite structure of the NPC symmetric core. The composite structure shown here, viewed from above the cytoplasmic face, was generated by means of sequential unbiased docking of nucleoporin and nucleoporin complex crystal structures into a previously reported cryo-ET reconstruction of the intact human NPC. Nucleoporin structures are shown as colored cartoons, and the nuclear envelope density is shown as a gray surface. The nuclear pore complex (NPC) controls the transport of macromolecules between the nucleus and cytoplasm, but its molecular architecture has thus far remained poorly defined. We biochemically reconstituted NPC core protomers and elucidated the underlying protein-protein interaction network. Flexible linker sequences, rather than interactions between the structured core scaffold nucleoporins, mediate the assembly of the inner ring complex and its attachment to the NPC coat. X-ray crystallographic analysis of these scaffold nucleoporins revealed the molecular details of their interactions with the flexible linker sequences and enabled construction of full-length atomic structures. By docking these structures into the cryoelectron tomographic reconstruction of the intact human NPC and validating their placement with our nucleoporin interactome, we built a composite structure of the NPC symmetric core that contains ~320,000 residues and accounts for ~56 megadaltons of the NPC’s structured mass. Our approach provides a paradigm for the structure determination of similarly complex macromolecular assemblies.