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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Thermodynamics and dynamics of histidine-binding protein, the water-soluble receptor of histidine permease. Implications for the transport of high and low affinity ligands.

The bacterial histidine permease is a model system for ABC transporters (traffic ATPases). The water-soluble receptor of this permease, HisJ, binds L-histidine and L-arginine (tightly) and L-lysine and L-ornithine (less tightly) in the periplasm, interacts with the membrane-bound complex (HisQMP2) and induces its ATPase activity, which results in ligand translocation. HisJ is a two-domain protein; in the absence of ligand, the cleft between two domains is open and binding of substrate stabilizes the closed conformation.

Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding.

An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized.

Structure of a monoclonal 2E8 Fab antibody fragment specific for the low-density lipoprotein-receptor binding region of apolipoprotein E refined at 1.9 A.

The crystal structure of the Fab fragment of 2E8, the monoclonal IgG1,kappa antibody specific for the low-density lipoprotein (LDL) receptor-binding region of apolipoprotein E (apoE), has been solved by molecular replacement and refined at 1.9 A resolution (PDB entry 12E8). Two 2E8 Fab molecules in the asymmetric unit are related by noncrystallographic symmetry and are hydrogen bonded through a beta-sheet-like intermolecular contact between the heavy-chain complementarity-determining regions 3 (CDRH3) of each molecule.

Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine.

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range.

Novel mechanism for defective receptor binding of apolipoprotein E2 in type III hyperlipoproteinemia.

The defective binding of apolipoprotein (apo) E2 to lipoprotein receptors, an underlying cause of type III hyperlipoproteinemia, results from replacement of Arg 158 with Cys, disrupting the naturally occurring salt bridge between Asp 154 and Arg 158. A new bond between Asp 154 and Arg 150 is formed, shifting Arg 150 out of the receptor binding region. Elimination of the 154-150 salt bridge by site-directed mutagenesis of Asp 154 to Ala restored the receptor binding activity to near normal levels.

2 A resolution structure of DppA, a periplasmic dipeptide transport/chemosensory receptor.

The family of about 50 periplasmic binding proteins, which exhibit diverse specificity (e.g., carbohydrates, amino acids, dipeptides, oligopeptides, oxyanions, metals, and vitamins) and range in size from 20 to 58 kDa, is a gold mine for an atomic-level investigation of structure and molecular recognition.

Influence of divalent cations in protein crystallization.

We have tested the effect of several cations in attempts to crystallize the ligand-bound forms of the leucine/isoleucine/valine-binding protein (LIVBP) (M(r) = 36,700) and leucine-specific binding protein (LBP) (M(r) = 37,000), which act as initial periplasmic receptors for the high-affinity osmotic-shock-sensitive active transport system in bacterial cells. Success was achieved with Cd2+ promoting the most dramatic improvement in crystal size, morphology, and diffraction quality. This comes about 15 years after the ligand-free proteins were crystallized.

Refined 1.89-A structure of the histidine-binding protein complexed with histidine and its relationship with many other active transport/chemosensory proteins.

The structure of the histidine-binding protein (HBP, M(r) = 26,100), involved solely in active transport, has been determined by the molecular replacement technique and refined to 1.89-A resolution and to an R-factor of 0.199.

Leucine/isoleucine/valine-binding protein contracts upon binding of ligand.

Small-angle x-ray scattering and computer modeling have been used to study the effects of ligand binding to the leucine/isoleucine/valine-binding protein, an initial component of the high-affinity active transport system for branched-chain aliphatic amino acids in Escherichia coli. Measurements were made with no ligand present and with either L-leucine or L-valine present. Upon binding of either leucine or valine, there is a decrease in the radius of gyration, from 23.

[Spatial structure of the basic chain of the neurotoxin I molecule from the venom of cobra Naja naja oxiana and its crystal packing].

The structure of the alpha-carbon chain was solved by molecular replacement method at 2.7 A resolution. Neurotoxin I (NTX-I) is one of the main protein components purified from the venom of the central asian cobra Naja naja oxiana.

Crystallization and preliminary x-ray diffraction study of neurotoxin-I from Naja naja oxiana venom.

Crystals of the neurotoxin-I (NTX-I) from the venom of the middle Asian cobra Naja naja oxiana have been grown by vapour diffusion and dialysis methods. The crystals belong to space group P2(1)2(1)2 with dimension of a = 25.19 A, b = 75.

Preliminary X-ray investigation of 70 S ribosome crystals from Thermus thermophilus.

Large three-dimensional crystals of 70 S from Thermus thermophilus have been grown from solutions of 2-methyl-2,4-pentanediol at 4 degrees C and examined in an X-ray synchrotron beam. The space group is P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = 510 A and c = 378 A. The diffraction patterns extend to better than 20 A.

Crystallization and preliminary X-ray crystallographic data of a histidine-binding protein from Escherichia coli.

Histidine-binding protein, purified from periplasmic space of Escherichia coli K12, has been crystallized in a form suitable for X-ray analysis. Crystals of average size 0.3 mm x 0.