Summary Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm, their large size and dynamic nature have impeded a full structural and functional elucidation. Here, we have determined a subnanometer precision structure for the entire 552-protein yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. The structure reveals the NPC’s functional elements in unprecedented detail. The NPC is built of sturdy diagonal columns to which are attached connector cables, imbuing both strength and flexibility, while tying together all other elements of the NPC, including membrane-interacting regions and RNA processing platforms. Inwardly-directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized in distinct functional units. Taken together, this integrative structure allows us to rationalize the architecture, transport mechanism, and evolutionary origins of the NPC.

Nanobodies are single domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show great promise as high affinity reagents for research, diagnostics and therapeutics due to their high specificity, small size (~15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Here, we present a rapid, straightforward approach that generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with Kd values into the sub-nanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultra-high affinity dimeric nanobodies with Kds down to ~30 pM. The approach presented is well-suited for the routine production of high affinity capture reagents for various biomedical applications.

Summary The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14 MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and mRNP remodeling machinery right over the NPC's central channel, rather than on distal cytoplasmic filaments as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

Highlights d A comprehensive model is presented of the yeast nuclear pore complex (NPC) d Connectors link together different structural and functional layers in the NPC d Multiple structural and functional NPC isoforms co-exist in each cell d Modular construction allows structural plasticity and inner ring dilation of the NPC

Type I Interferons (IFNs) are a family of homologous helical cytokines initiating strong anti-viral and anti-proliferative activity. All type I IFNs bind to a common cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. We studied intermolecular interactions between IFNAR2-EC and IFNα2 using asymmetric reverse-protonation of the different complex components and 2D homonuclear NOESY. This new approach revealed with excellent signal-to-noise ratio 24 new intermolecular NOEs between the two molecules despite the low concentration of the complex (0.25 mM) and its high molecular weight (44 kDA). Sequential and side-chain assignment of IFNAR2-EC and IFNα2 in their binary complex helped assign the inter-molecular NOEs to the corresponding protons. A docking model of the IFNAR2-EC/IFNα2 complex was calculated based on the inter-molecular interactions found in the present study as well as four double mutant cycle constraints, previously observed NOEs between a single pair of residues and the NMR mapping of the binding sites on IFNAR2-EC and IFNα2. Our docking model doubles the buried surface area of the previous model and significantly increases the number of inter-molecular hydrogen bonds, salt bridges and Van der-Waals interactions. Furthermore, the current model reveals participation of several new regions in the binding site such as the N-terminus and A-helix of IFNα2 and the C-domain of IFNAR2-EC. As a result of these additions, the orientation of IFNAR2-EC relative to IFNα2 has changed by 30° in comparison with a previously calculated model that was based on NMR mapping of the binding sites and double mutant cycle constraints. In addition, the new model strongly supports the recently proposed allosteric changes in IFNα2 upon IFNAR1-EC binding to the binary IFNα2/ IFNAR2-EC complex.Type I IFNs are a major component of the innate immune system protecting against viral infection. They provide an early line of defense, hours to days ahead of the adaptive immune † This study was supported by the Israel Science Foundation, NIH Grant GM53329 and the Kimmelman Center. J.A. is the Dr. Joseph and Ruth Owades Professor of Chemistry. * To whom correspondence should be addressed. Jacob. . NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 June 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript response, and are essential for the survival of higher vertebrates (1-3). In addition to a strong anti-viral activity, type I IFNs are also attributed anti-proliferative and immunomodulatory properties (4,5).All human type I IFNs elicit their activity through the same cell surface receptor consisting of two trans-membranal protein subunits, IFNAR1 and IFNAR2 (6,7). IFNAR2 is the major ligand binding component and has nM affinity to IFNs without the presence of IFNAR1. The affinity of the IFNAR1 subunit to IFNs is much lower and the dissociation constant is in the μM range (8). The IFN signaling process begins ...