Ferdinand M. Huber

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.

Structural and Functional Analysis of Human SIRT1

SIRT1 is a NAD(+)-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD(+)-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD(+)-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.

Architecture of the fungal nuclear pore inner ring complex

Building a gate to the nucleus Nuclear pore complexes form a gateway between the cytoplasm and the nucleus (see the Perspective by Ullman and Powers). Stuwe et al. combined structural, biochemical, and functional analyses to elucidate the architecture of a six-protein complex that makes up the inner ring of the fungal nuclear pore. This includes a central trimeric complex homologous to the Nup62 complex found in metazoans that is incorporated into the nuclear pore inner-ring complex. Chug et al. report the structure of the metazoan trimeric Nup62 complex. Neither study supports a model in which the pore can dilate and constrict. Instead they suggest a rigid pore in which flexible domains called FG repeats fill the channel and form a barrier that can be traversed by receptors that carry cargos across. Science, this issue pp. 56 and 106; see also p. 33 A central complex in the nuclear pore forms a rigid channel filled with flexible domains that form a barrier. [Also see Perspective by Ullman and Powers] The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.

A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins

Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.19105.001

Molecular basis for protection of ribosomal protein L4 from cellular degradation

Eukaryotic ribosome biogenesis requires the nuclear import of ∼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery.

Histone-binding of DPF2 mediates its repressive role in myeloid differentiation

Significance Double plant homeodomain finger 2 (DPF2) is a regulator of myeloid differentiation and implicated in a range of human cancers, including acute myelogenous leukemia. Recruitment of DPF2 to chromatin has been shown to alter the expression of target genes and inhibit myeloid differentiation. Here, we present the crystal structure of the human DPF2 tandem plant homeodomain finger domain and comprehensive structure-guided biochemical and in vivo analyses. Combined, our data delineate the determinants of DPF2’s chromatin recruitment and establish its regulatory role in human hematopoietic stem/progenitor cell differentiation. Double plant homeodomain finger 2 (DPF2) is a highly evolutionarily conserved member of the d4 protein family that is ubiquitously expressed in human tissues and was recently shown to inhibit the myeloid differentiation of hematopoietic stem/progenitor and acute myelogenous leukemia cells. Here, we present the crystal structure of the tandem plant homeodomain finger domain of human DPF2 at 1.6-Å resolution. We show that DPF2 interacts with the acetylated tails of both histones 3 and 4 via bipartite binding pockets on the DPF2 surface. Blocking these interactions through targeted mutagenesis of DPF2 abolishes its recruitment to target chromatin regions as well as its ability to prevent myeloid differentiation in vivo. Our findings suggest that the histone binding of DPF2 plays an important regulatory role in the transcriptional program that drives myeloid differentiation.