A checkpoint function for Nup98 in nuclear pore formation suggested by novel inhibitory nanobodies.

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from frog to human. We also report a simplified assay that directly tracks postmitotic NPC assembly with added fluorophore-labeled anti-Nup nanobodies. During interphase, NPCs are inserted into a pre-existing nuclear envelope. Monitoring this process is challenging because newly assembled NPCs are indistinguishable from pre-existing ones. We overcame this problem by inserting Xenopus-derived NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We further asked whether anti-Nup nanobodies could serve as NPC assembly inhibitors. Using a selection strategy against conserved epitopes, we obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93 α-solenoid domain in recruiting Nup358 and the Nup214·88·62 complex, as well as for Nup155 and the Nup98 autoproteolytic domain in NPC scaffold assembly. The latter suggests a checkpoint linking pore formation to the assembly of the Nup98-dominated permeability barrier.

Structural basis for the nuclear import and export functions of the biportin Pdr6/Kap122.

Importins ferry proteins into nuclei while exportins carry cargoes to the cytoplasm. In the accompanying paper in this issue (Vera Rodriguez et al. 2019. J. Cell Biol. https://doi.org/10.1083/jcb.201812091), we discovered that Pdr6 is a biportin that imports, e.g., the SUMO E2 ligase Ubc9 while depleting the translation factor eIF5A from the nuclear compartment. In this paper, we report the structures of key transport intermediates, namely, of the Ubc9•Pdr6 import complex, of the RanGTP•Pdr6 heterodimer, and of the trimeric RanGTP•Pdr6•eIF5A export complex. These revealed nonlinear transport signals, chaperone-like interactions, and how the RanGTPase system drives Pdr6 to transport Ubc9 and eIF5A in opposite directions. The structures also provide unexpected insights into the evolution of transport selectivity. Specifically, they show that recognition of Ubc9 by Pdr6 differs fundamentally from that of the human Ubc9-importer Importin 13. Likewise, Pdr6 recognizes eIF5A in a nonhomologous manner compared with the mammalian eIF5A-exporter Exportin 4. This suggests that the import of Ubc9 and active nuclear exclusion of eIF5A evolved in different eukaryotic lineages more than once and independently from each other.

Structure of the exportin Xpo4 in complex with RanGTP and the hypusine-containing translation factor eIF5A.

Xpo4 is a bidirectional nuclear transport receptor that mediates nuclear export of eIF5A and Smad3 as well as import of Sox2 and SRY. How Xpo4 recognizes such a variety of cargoes is as yet unknown. Here we present the crystal structure of the RanGTP·Xpo4·eIF5A export complex at 3.2 Å resolution. Xpo4 has a similar structure as CRM1, but the NES-binding site is occluded, and a new interaction site evolved that recognizes both globular domains of eIF5A. eIF5A contains hypusine, a unique amino acid with two positive charges, which is essential for cell viability and eIF5A function in translation. The hypusine docks into a deep, acidic pocket of Xpo4 and is thus a critical element of eIF5A's complex export signature. This further suggests that Xpo4 recognizes other cargoes differently, and illustrates how Xpo4 suppresses - in a chaperone-like manner - undesired interactions of eIF5A inside nuclei.

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation.

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks ­ each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.

Crystal structure of the metazoan Nup62•Nup58•Nup54 nucleoporin complex.

Nuclear pore complexes (NPCs) conduct nucleocytoplasmic transport and gain transport selectivity through nucleoporin FG domains. Here, we report a structural analysis of the FG Nup62•58•54 complex, which is a crucial component of the transport system. It comprises a ≈13 nanometer-long trimerization interface with an unusual 2W3F coil, a canonical heterotrimeric coiled coil, and a kink that enforces a compact six-helix bundle. Nup54 also contains a ferredoxin-like domain. We further identified a heterotrimeric Nup93-binding module for NPC anchorage. The quaternary structure alternations in the Nup62 complex, which were previously proposed to trigger a general gating of the NPC, are incompatible with the trimer structure. We suggest that the highly elongated Nup62 complex projects barrier-forming FG repeats far into the central NPC channel, supporting a barrier that guards the entire cross section.

Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome.

The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 Å crystal structure of the RF3·GDPNP·ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7° rotation of the body and 14° rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.

Crystal structure of a translation termination complex formed with release factor RF2.

We report the crystal structure of a translation termination complex formed by the Thermus thermophilus 70S ribosome bound with release factor RF2, in response to a UAA stop codon, solved at 3 A resolution. The backbone of helix alpha5 and the side chain of serine of the conserved SPF motif of RF2 recognize U1 and A2 of the stop codon, respectively. A3 is unstacked from the first 2 bases, contacting Thr-216 and Val-203 of RF2 and stacking on G530 of 16S rRNA. The structure of the RF2 complex supports our previous proposal that conformational changes in the ribosome in response to recognition of the stop codon stabilize rearrangement of the switch loop of the release factor, resulting in docking of the universally conserved GGQ motif in the PTC of the 50S subunit. As seen for the RF1 complex, the main-chain amide nitrogen of glutamine in the GGQ motif is positioned to contribute directly to catalysis of peptidyl-tRNA hydrolysis, consistent with mutational studies, which show that most side-chain substitutions of the conserved glutamine have little effect. We show that when the H-bonding capability of the main-chain N-H of the conserved glutamine is eliminated by substitution with proline, peptidyl-tRNA esterase activity is abolished, consistent with its proposed role in catalysis.

Structural basis for translation termination on the 70S ribosome.

At termination of protein synthesis, type I release factors promote hydrolysis of the peptidyl-transfer RNA linkage in response to recognition of a stop codon. Here we describe the crystal structure of the Thermus thermophilus 70S ribosome in complex with the release factor RF1, tRNA and a messenger RNA containing a UAA stop codon, at 3.2 A resolution. The stop codon is recognized in a pocket formed by conserved elements of RF1, including its PxT recognition motif, and 16S ribosomal RNA. The codon and the 30S subunit A site undergo an induced fit that results in stabilization of a conformation of RF1 that promotes its interaction with the peptidyl transferase centre. Unexpectedly, the main-chain amide group of Gln 230 in the universally conserved GGQ motif of the factor is positioned to contribute directly to peptidyl-tRNA hydrolysis.

Interactions and dynamics of the Shine Dalgarno helix in the 70S ribosome.

The crystal structure of an initiation-like 70S ribosome complex containing an 8-bp Shine-Dalgarno (SD) helix was determined at 3.8-A resolution. Translation-libration-screw analysis showed that the inherent anisotropic motions of the SD helix were biased along its helical axis, suggesting that during the first step of translocation, the SD helix moves along its helical screw axis. Contacts between the SD helix and the ribosome were primarily through interactions with helices 23a, 26, and 28 of 16S rRNA. Contact with the neck (helix 28) of the 30S subunit near its hinge point suggests that formation of the SD helix could affect positioning of the head of the 30S subunit for optimal interaction with initiator tRNA. The bulged U723 in helix 23a interacts with the minor groove of the SD helix at the C1539.G-10 base pair, explaining its selective conservation in bacteria and archaea.

Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements.

Our understanding of the mechanism of protein synthesis has undergone rapid progress in recent years as a result of low-resolution X-ray and cryo-EM structures of ribosome functional complexes and high-resolution structures of ribosomal subunits and vacant ribosomes. Here, we present the crystal structure of the Thermus thermophilus 70S ribosome containing a model mRNA and two tRNAs at 3.7 A resolution. Many structural details of the interactions between the ribosome, tRNA, and mRNA in the P and E sites and the ways in which tRNA structure is distorted by its interactions with the ribosome are seen. Differences between the conformations of vacant and tRNA-bound 70S ribosomes suggest an induced fit of the ribosome structure in response to tRNA binding, including significant changes in the peptidyl-transferase catalytic site.

Ligand-free and -bound structures of the binding protein (LivJ) of the Escherichia coli ABC leucine/isoleucine/valine transport system: trajectory and dynamics of the interdomain rotation and ligand specificity.

The leucine/isoleucine/valine-binding protein (LIVBP or LivJ) serves as the primary high-affinity receptor of the Escherichia coli ABC-type transporter for the three aliphatic amino acids. The first structure of LIVBP determined previously without bound ligand showed a molecule comprised of two domains which are far apart and bisected by a wide open, solvent-accessible cleft. Here we report the crystal structures of another ligand-free state and three complexes with the aliphatic amino acids. In the present ligand-free structure, the two domains are farther apart. In the three very similar complex structures, the two domains are in close proximity, and each desolvated ligand is completely engulfed in the cleft and bound by both domains. The two different ligand-free structures, combined with those of the very similar ligand-bound structures, indicate the trajectory and backbone torsion angle changes of the hinges that accompany domain closure and play crucial functional roles. The amino acids are bound by polar and nonpolar interactions, occurring predominantly in one domain. Consistent with the protein specificity, the aliphatic side chains of the ligands lie in a hydrophobic pocket fully formed following domain or cleft closure. Comparison of the structures of LIVBP with several different binding proteins indicates no correlations between the magnitudes of the hinge-bending angles and the protein masses, the ligand sizes, or the number of segments connecting the two domains. Results of normal-mode analysis and molecular dynamics simulations are consistent with the trajectory and intrinsic flexibility of the interdomain hinges and the dominance of one domain in ligand binding in the open state.