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.

The nuclear pore complex (NPC) serves as the sole bidirectional gateway of macromolecules in and out of the nucleus. Owing to its size and complexity (~1,000 protein subunits, ~110 MDa in humans), the NPC has remained one of the foremost challenges for structure determination. Structural studies have now provided atomic-resolution crystal structures of most nucleoporins. The acquisition of these structures, combined with biochemical reconstitution experiments, cross-linking mass spectrometry, and cryo–electron tomography, has facilitated the determination of the near-atomic overall architecture of the symmetric core of the human, fungal, and algal NPCs. Here, we discuss the insights gained from these new advances and outstanding issues regarding NPC structure and function. The powerful combination of bottom-up and top-down approaches toward determining the structure of the NPC offers a paradigm for uncovering the architectures of other complex biological machines to near-atomic resolution.

Introduction The nuclear pore complex (NPC) is the primary gateway for transport of macromolecules between the nucleus and cytoplasm, serving as both a critical mediator and regulator of gene expression. NPCs are enormous ~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 cryoelectron tomography and in determining atomic resolution crystal structures of nucleoporins, the molecular architecture of the assembled NPC remains poorly understood, hindering the design of mechanistic studies that could investigate its many roles in cell biology. Rationale Existing cryoelectron tomographic reconstructions of the NPC remain 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. Lastly, by superposing crystal structures that overlapped in sequence, we generated accurate full-length structures of the large scaffold nucleoporins. Supported by this biochemical data, we used sequential, unbiased searches to place the nucleoporin crystal structures into a previously determined cryoelectron tomographic 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 utilize disparate mechanisms of interaction. While 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 with limited cross-spoke interactions. Most nucleoporins are present in 32 copies, with notable exceptions of Nup170 and Nup188. Lastly, we observed the arrangement of the channel nucleoporins, which orient their N-termini into two sixteen-membered rings, 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 probe 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 unique genes. Our integrated, bottom-up approach provides a paradigm for the biochemical and structural characterization of similarly large biological mega-assemb...

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.

The reversible methylation of specific lysine residues in histone tails is crucial in epigenetic gene regulation. LSD1, the first known lysine-specific demethylase, selectively removes monomethyl and dimethyl, but not trimethyl modifications of Lys4 or Lys9 of histone-3. Here, we present the crystal structure of LSD1 at 2.9-A resolution. LSD1 forms a highly asymmetric, closely packed domain structure from which a long helical 'tower' domain protrudes. The active site cavity is spacious enough to accommodate several residues of the histone tail substrate, but does not appear capable of recognizing the different methylation states of the substrate lysine. This supports the hypothesis that trimethylated lysine is chemically rather than sterically discriminated. We present a biochemical analysis of LSD1 mutants that identifies crucial residues in the active site cavity and shows the importance of the SWIRM and tower domains for catalysis.