Structures of the tRNA export factor in the nuclear and cytosolic states.

Transfer RNAs are among the most ubiquitous molecules in cells, central to decoding information from messenger RNAs on translating ribosomes. In eukaryotic cells, tRNAs are actively transported from their site of synthesis in the nucleus to their site of function in the cytosol. This is mediated by a dedicated nucleo-cytoplasmic transport factor of the karyopherin-beta family (Xpot, also known as Los1 in Saccharomyces cerevisiae). Here we report the 3.2 A resolution structure of Schizosaccharomyces pombe Xpot in complex with tRNA and RanGTP, and the 3.1 A structure of unbound Xpot, revealing both nuclear and cytosolic snapshots of this transport factor. Xpot undergoes a large conformational change on binding cargo, wrapping around the tRNA and, in particular, binding to the tRNA 5' and 3' ends. The binding mode explains how Xpot can recognize all mature tRNAs in the cell and yet distinguish them from those that have not been properly processed, thus coupling tRNA export to quality control.

Structural and functional analysis of slit and heparin binding to immunoglobulin-like domains 1 and 2 of Drosophila Robo.

Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.

A molecular mechanism for the heparan sulfate dependence of slit-robo signaling.

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.

SMG7 is a 14-3-3-like adaptor in the nonsense-mediated mRNA decay pathway.

In metazoa, regulation of the phosphorylation state of UPF1 is crucial for nonsense-mediated mRNA decay (NMD), a process by which aberrant mRNAs containing nonsense mutations are degraded. UPF1 is targeted for dephosphorylation by three related proteins, SMG5, SMG6, and SMG7. We report here the crystal structure of the N-terminal domain of SMG7. The structure reveals that SMG7 contains a 14-3-3-like domain. Residues that bind phosphoserine-containing peptides in 14-3-3 are conserved at the equivalent positions in SMG7. Mutation of these residues impairs UPF1 binding to SMG7 in vitro and UPF1 recruitment to cytoplasmic mRNA decay foci in vivo, suggesting that SMG7 acts as an adaptor in targeting mRNAs associated with phosphorylated UPF1 for degradation. The 14-3-3 site of SMG7 is conserved in SMG5 and SMG6. These data also imply that the homologous human Est1 might have a 14-3-3 function at telomeres, and that phosphorylation events may be important for telomerase regulation.

Conformational variability of nucleo-cytoplasmic transport factors.

The transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is largely mediated by a single family of transport factors, the karyopherin or importin beta-like family. Structural and biochemical evidence suggests conformational flexibility of these modular HEAT-repeat proteins is crucial for their regulation. Here we use small angle x-ray scattering to assess the extent of conformational variation within a set of nuclear import and export factors. The study reveals that importin beta, transportin, and the exportin Xpo-t share a similar S-like superhelical conformation in their unbound state. There are no obvious differences in the overall structures that might generally distinguish nuclear export from nuclear import mediators. Two other members of the family, the exportins Cse1 and Xpo1, possess a significantly more globular conformation, indicating that the extended S-like architecture is not a hallmark of all karyopherins. Binding of RanGTP/cargo to importin beta, transportin, and Xpo-t triggers distinct conformational responses, suggesting that even closely related karyopherins employ different mechanisms of conformational regulation and that cargo and nuclear pore-interacting surfaces of the different receptors may be unique.