Chaetomium Thermophilum Research Articles

Translation Initiation Factor eIF3b Contains a Nine-Bladed β-Propeller and Interacts with the 40S Ribosomal Subunit

The multisubunit eukaryotic translation initiation factor 3, among which the subunit b (eIF3b) is a major scaffold protein, plays essential roles in protein synthesis. Here, we report the crystal structure of the WD40 domain of Chaetomium thermophilum eIF3b, revealing a nine-bladed β-propeller fold. Sequence analysis indicates that this propeller architecture is common to all eIF3b orthologs. Revisiting the cryoelectron microscopy (cryo-EM) map of the 43S preinitiation complex suggests an interaction of the eIF3b with the 40S ribosomal subunit involving the ribosomal protein S9e and the 18S rRNA. This model is strongly supported by the direct binding of eIF3b to 40S ribosomes and to the isolated ribosomal protein rpS9e in vitro.

A Novel β-Glucosidase from Humicola insolens with High Potential for Untreated Waste Paper Conversion to Sugars

Humicola insolens produced a new β-glucosidase (BglHi2) under solid-state fermentation. The purified enzyme showed apparent molecular masses of 116 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and 404 kDa (gel-filtration), suggesting that it is a homotetramer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Chaetomium thermophilum. Optima of pH and temperature were 5.0 and 65 °C, respectively, and the enzyme was stable for 60 min at 50 °C, maintaining 71 % residual activity after 60 min at 55 °C. BglHi2 hydrolyzed p-nitrophenyl-β-D-glucopyranoside and cellobiose. Cellobiose hydrolysis occurred with high apparent affinity (K M = 0.24 ± 0.01 mmol L(-1)) and catalytic efficiency (k cat/K M = 1,304.92 ± 53.32 L mmol(-1) s(-1)). The activity was insensitive to Fe(+3), Cr(+2), Mn(+2), Co(+2), and Ni(2+), and 50-60 % residual activities were retained in the presence of Pb(2+), Hg(2+), and Cu(2+). Mixtures of pure BglHi2 or H. insolens crude extract (CE) with crude extracts from Trichoderma reesei fully hydrolyzed Whatman no. 1 paper. Mixtures of H. insolens CE with T. reesei CE or Celluclast 1.5 L fully hydrolyzed untreated printed office paper, napkin, and magazine papers after 24-48 h, and untreated cardboard was hydrolyzed by a H. insolens CE/T. reesei CE mixture with 100 % glucose yield. Data revealed the good potential of BglHi2 for the hydrolysis of waste papers, promising feedstocks for cellulosic ethanol production.

A homing endonuclease with a switch: Characterization of a twintron encoded homing endonuclease

The small ribosomal subunit gene residing in the mitochondrial DNA of the thermophilic fungus Chaetomium thermophilum var. thermophilum La Touche DSM 1495 is interrupted by a twintron at position mS1247. The mS1247 twintron represents the first mixed twintron found in fungal mtDNA, composed of an external group I intron encoding a LAGLIDADG open reading frame that is interrupted by an internal group II intron. Splicing of the internal group II intron reconstitutes the open reading frame and thus facilitates the expression of the encoded homing endonuclease. The cleavage assays suggest that the twintron encodes an active homing endonuclease that could potentially mobilize the twintron to rns genes that have not yet been invaded by this mobile composite element.

Crystal structure and biochemical characterization of a manganese superoxide dismutase from Chaetomium thermophilum

A manganese superoxide dismutase from the thermophilic fungus Chaetomium thermophilum (CtMnSOD) was expressed in Pichia pastoris and purified to homogeneity. Its optimal temperature was 60°C with approximately 75% of its activity retained after incubation at 70°C for 60min. Recombinant yeast cells carrying C. thermophilum mnsod gene exhibited higher stress resistance to salt and oxidative stress-inducing agents than control yeast cells. In an effort to provide structural insights, CtMnSOD was crystallized and its structure was determined at 2.0Å resolution. The overall architecture of CtMnSOD was found similar to other MnSODs with highest structural similarities obtained against a MnSOD from the thermotolerant fungus Aspergillus fumigatus. In order to explain its thermostability, structural and sequence analysis of CtMnSOD with other MnSODs was carried out. An increased number of charged residues and an increase in the number of intersubunit salt bridges and the Thr:Ser ratio were identified as potential reasons for the thermostability of CtMnSOD.

Structural basis for the molecular recognition of polyadenosine RNA by Nab2 Zn fingers

The yeast poly(A) RNA binding protein, Nab2, facilitates poly(A) tail length regulation together with targeting transcripts to nuclear pores and their export to the cytoplasm. Nab2 binds polyadenosine RNA primarily through a tandem repeat of CCCH Zn fingers. We report here the 2.15 Å resolution crystal structure of Zn fingers 3–5 of Chaetomium thermophilum Nab2 bound to polyadenosine RNA and establish the structural basis for the molecular recognition of adenosine ribonucleotides. Zn fingers 3 and 5 each bind two adenines, whereas finger 4 binds only one. In each case, the purine ring binds in a surface groove, where it stacks against an aromatic side chain, with specificity being provided by a novel pattern of H-bonds, most commonly between purine N6 and a Zn-coordinated cysteine supplemented by H-bonds between purine N7 and backbone amides. Residues critical for adenine binding are conserved between species and provide a code that allows prediction of finger-binding stoichiometry based on their sequence. Moreover, these results indicate that, in addition to poly(A) tails, Nab2 can also recognize sequence motifs elsewhere in transcripts in which adenosines are placed at key positions, consistent with its function in mRNP organization and compaction as well as poly(A) tail length regulation.

Protein Interfaces of the Conserved Nup84 Complex from Chaetomium thermophilum Shown by Crosslinking Mass Spectrometry and Electron Microscopy

A key building block of the nuclear pore complex (NPC) is the Nup84 subcomplex that has been structurally analyzed predominantly in the yeast system. To expand this analysis and gain insight into the evolutionary conservation of its structure, we reconstituted an octameric Nup84 complex using the subunits from a thermophile, Chaetomium thermophilum (ct). This assembly carries Nup37 and Elys, which are characteristic subunits of the orthologous human Nup107-Nup160 complex but absent from the yeast Saccharomyces cerevisiae. We found that Elys binds cooperatively to the complex requiring both Nup37 and Nup120. Unexpectedly, the reconstituted ctNup84 complex formed a striking dimer structure with an unpredicted side-to-side arrangement of two molecules. Finally, crosslinking mass spectrometry allowed the mapping of key protein interfaces within the Y-shaped complex. Thus, the thermophilic Nup84 complex can serve as a structural model for higher eukaryotic Nup107-Nup160 assemblies to gain insight into its possible configuration within the NPC scaffold.

Structural basis of histone H2A–H2B recognition by the essential chaperone FACT

Facilitates chromatin transcription (FACT) is a conserved histone chaperone that reorganizes nucleosomes and ensures chromatin integrity during DNA transcription, replication and repair. Key to the broad functions of FACT is its recognition of histones H2A-H2B (ref. 2). However, the structural basis for how histones H2A-H2B are recognized and how this integrates with the other functions of FACT, including the recognition of histones H3-H4 and other nuclear factors, is unknown. Here we reveal the crystal structure of the evolutionarily conserved FACT chaperone domain Spt16M from Chaetomium thermophilum, in complex with the H2A-H2B heterodimer. A novel 'U-turn' motif scaffolded onto a Rtt106-like module embraces the α1 helix of H2B. Biochemical and in vivo assays validate the structure and dissect the contribution of histone tails and H3-H4 towards Spt16M binding. Furthermore, we report the structure of the FACT heterodimerization domain that connects FACT to replicative polymerases. Our results show that Spt16M makes several interactions with histones, which we suggest allow the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA. FACT would thus enhance 'nucleosome breathing' by re-organizing the first 30 base pairs of nucleosomal histone-DNA contacts. Our snapshot of the engagement of the chaperone with H2A-H2B and the structures of all globular FACT domains enable the high-resolution analysis of the vital chaperoning functions of FACT, shedding light on how the complex promotes the activity of enzymes that require nucleosome reorganization.

Purification, bio-chemical characterization, homology modeling and active site binding mode interactions of thermo-alkali-tolerant β-1,4 endoxylanase from Coprinus cinereus LK-D-NCIM-1369

Abstract Present study aims at purifying and characterizing crude xylanase isolated from Coprinus cinereus LK-D-NCIM-1369 under solid-state fermentation conditions. Crude xylanase is purified by (NH4) 2 SO 4 precipitation, carboxymethyl cellulose cation-exchange and gel filtration chromatography with Superdex-200 column. Purified xylanase from C. cinereus shows 54.3% similarity with β-1,4 endoxylanase from C. cinerea okayama7#130 with accession number gi|169855830 based on MALDI-TOF/TOF. The purified xylanase—a monomeric protein with molecular weight 20.1 kD shows maximum activity at 60 °C and pH 7.0 and stable over pH 5.0–9.0 and temperature up to 70 °C. The xylanase shows K m and V values of 3.26 mg/mL and 909.09 µm/min/mg for birchwood xylan. A sequence of 186 amino acids of C. cinerea okayama7#130 gi|169855830 is retrieved from NCBI database and its 3-D model is generated on the basis of crystal structure of xylanase 1XNK-A from Chaetomium thermophilum with the help of online server SWISS-MODEL workspace. The model is verified and validated on SAVES and PROCHECK programmes, respectively. Ramachandran plot reveals that the total residues in allowed and generously allowed regions are 99.4% and 0.6% respectively. Overlapping of xylanase with the template of 1XNK-A stipulates the amino acid residues Asn35, Tyr68, Arg113, Ser118, Tyr168 and Glu174 constitute active site of the enzyme.

Structure of the eukaryotic atypical RIO kinases reveals ATPase‐dependent mechanism in ribosome biogenesis

RIO kinases are ancient atypical protein kinases essential for the final maturation step of the small subunit of the ribosome. The crystal structures of two eukaryotic RIO kinases, Rio2 from Chaetomium thermophilum and human RioK1, in the presence of ATP, were determined to 2.5 and 2.8 Å respectively. Unexpectedly, a novel phosphoaspartate intermediate was observed in both structures. Acyl‐phosphate intermediates, observed in P‐type ATPases, are unusual for serine protein kinases. Prompted by this new information, we show that RIO kinases have significant ATPase activity. Additionally, docking of the eukaryotic Rio2 kinase into a reported cryo‐electron microscopy map of the pre‐40S particles and yeast genetic studies show that although catalytic activity is required for function, the active site is inaccessible when the RIO domain is bound to the pre‐ribosome. A new model for the functional mechanism of the RIO kinases is therefore suggested that includes a role for a transient intermediate and ATPase activity in pre‐40S maturation. We propose that RIO kinases function not as protein kinases, but as ATPases that sense pre‐40S conformational change and drive particles to maturation. These data collectively provide surprising new insight into the role of RIO kinases in ribosome biogenesis.

The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro

The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.

Deep transcriptome-sequencing and proteome analysis of the hydrothermal vent annelid Alvinella pompejana identifies the CvP-bias as a robust measure of eukaryotic thermostability

BackgroundAlvinella pompejana is an annelid worm that inhabits deep-sea hydrothermal vent sites in the Pacific Ocean. Living at a depth of approximately 2500 meters, these worms experience extreme environmental conditions, including high temperature and pressure as well as high levels of sulfide and heavy metals. A. pompejana is one of the most thermotolerant metazoans, making this animal a subject of great interest for studies of eukaryotic thermoadaptation.ResultsIn order to complement existing EST resources we performed deep sequencing of the A. pompejana transcriptome. We identified several thousand novel protein-coding transcripts, nearly doubling the sequence data for this annelid. We then performed an extensive survey of previously established prokaryotic thermoadaptation measures to search for global signals of thermoadaptation in A. pompejana in comparison with mesophilic eukaryotes. In an orthologous set of 457 proteins, we found that the best indicator of thermoadaptation was the difference in frequency of charged versus polar residues (CvP-bias), which was highest in A. pompejana. CvP-bias robustly distinguished prokaryotic thermophiles from prokaryotic mesophiles, as well as the thermophilic fungus Chaetomium thermophilum from mesophilic eukaryotes. Experimental values for thermophilic proteins supported higher CvP-bias as a measure of thermal stability when compared to their mesophilic orthologs. Proteome-wide mean CvP-bias also correlated with the body temperatures of homeothermic birds and mammals.ConclusionsOur work extends the transcriptome resources for A. pompejana and identifies the CvP-bias as a robust and widely applicable measure of eukaryotic thermoadaptation.ReviewerThis article was reviewed by Sándor Pongor, L. Aravind and Anthony M. Poole.

Consistent mutational paths predict eukaryotic thermostability

BackgroundProteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published.ResultsStudying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1), we could also characterise the molecular consequences of some of these mutations.ConclusionsThe comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.

Product inhibition of cellulases studied with 14C-labeled cellulose substrates

BackgroundAs a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched. To be economically feasible, the hydrolysis of lignocellulose must be conducted at a high concentration of solids, which results in high concentrations of hydrolysis end-products, cellobiose and glucose, making the relief of product inhibition of cellulases a major challenge in the process. However, little quantitative information on the product inhibition of individual cellulases acting on cellulose substrates is available because it is experimentally difficult to assess the hydrolysis of the heterogeneous polymeric substrate in the high background of added products.ResultsThe cellobiose and glucose inhibition of thermostable cellulases from Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum acting on uniformly 14C-labeled bacterial cellulose and its derivatives, 14C-bacterial microcrystalline cellulose and 14C-amorphous cellulose, was studied. Cellulases from Trichoderma reesei were used for comparison. The enzymes most sensitive to cellobiose inhibition were glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs), followed by family 6 CBHs and endoglucanases (EGs). The strength of glucose inhibition followed the same order. The product inhibition of all enzymes was relieved at higher temperatures. The inhibition strength measured for GH7 CBHs with low molecular-weight model substrates did not correlate with that measured with 14C-cellulose substrates.ConclusionsGH7 CBHs are the primary targets for product inhibition of the synergistic hydrolysis of cellulose. The inhibition must be studied on cellulose substrates instead of on low molecular-weight model substrates when selecting enzymes for lignocellulose hydrolysis. The advantages of using higher temperatures are an increase in the catalytic efficiency of enzymes and the relief of product inhibition.

Structure of the catalytic core module of theChaetomium thermophilumfamily GH6 cellobiohydrolase Cel6A

Cellulases, including cellobiohydrolases and endoglucanases, are important enzymes involved in the breakdown of the polysaccharide cellulose. These catalysts have found widescale industrial applications, particularly in the paper and textile industries, and are now finding use in `second-generation' conversion of biomass to biofuels. Despite this considerable biotechnological application, and undoubted future potential, uncertainty remains as to the exact reaction mechanism of the inverting cellulases found in the GH6 family of carbohydrate-active enzymes. In order to gain additional understanding as to how these societally beneficial biocatalysts function, the crystal structure of a GH6 cellobiohydrolase from Chaetomium thermophilum, CtCel6A, has been solved. This structure reveals a distorted α/β-barrel fold comprising a buried tunnel-like active site quite typical of Cel6A enzymes. Analysis of an enzyme-product complex (cellobiose in the -3 and -2 subsites and cellotetraose in subsites +1 to +4) supports the hypothesis that this group of enzymes act via an atypical single-displacement mechanism. Of particular note in this analysis is an active-centre metal ion, Li(+), the position of which matches the position of the positively charged anomeric carbon of the oxocarbenium-ion-like transition state.

Phylogenetic Analysis of the Complete Mitochondrial Genome of Madurella mycetomatis Confirms Its Taxonomic Position within the Order Sordariales

Background Madurella mycetomatis is the most common cause of human eumycetoma. The genus Madurella has been characterized by overall sterility on mycological media. Due to this sterility and the absence of other reliable morphological and ultrastructural characters, the taxonomic classification of Madurella has long been a challenge. Mitochondria are of monophyletic origin and mitochondrial genomes have been proven to be useful in phylogenetic analyses.ResultsThe first complete mitochondrial DNA genome of a mycetoma-causative agent was sequenced using 454 sequencing. The mitochondrial genome of M. mycetomatis is a circular DNA molecule with a size of 45,590 bp, encoding for the small and the large subunit rRNAs, 27 tRNAs, 11 genes encoding subunits of respiratory chain complexes, 2 ATP synthase subunits, 5 hypothetical proteins, 6 intronic proteins including the ribosomal protein rps3. In phylogenetic analyses using amino acid sequences of the proteins involved in respiratory chain complexes and the 2 ATP synthases it appeared that M. mycetomatis clustered together with members of the order Sordariales and that it was most closely related to Chaetomium thermophilum. Analyses of the gene order showed that within the order Sordariales a similar gene order is found. Furthermore also the tRNA order seemed mostly conserved.ConclusionPhylogenetic analyses of fungal mitochondrial genomes confirmed that M. mycetomatis belongs to the order of Sordariales and that it was most closely related to Chaetomium thermophilum, with which it also shared a comparable gene and tRNA order.

Comparative study for the kinetics of extracellular xylanases from Trichoderma harzianum and Chaetomium thermophilum

Xylanases assume special importance in the paper and pulp industry as they replace toxic chemicals such as chlorine and chlorine dioxide for developing eco-friendly processes. This study evaluated xylanases produced by two fungi, the mesophilic fungus Trichoderma harzianum and a thermophilic fungus Chaetomium thermophilum . Among the polymeric substrates studied for xylanase production by both the fungi, birch wood xylan was found to be the best inducer of xylanases. Xylanases induction was subject to glucose repression. Partially purified xylanases preparation from T. harzianum and C. thermophilum exhibited optimal activities at pH 5 and 6 and at 60oC and 70oC, respectively. The apparent K m and V max values for the partially purified xylanase from T. harzianum using oat spelt xylan as a substrate were 4.8 mg mL -1 and 0.526 µmol min -1 mg -1 , respectively. Whereas values of the partially purified xylanase from C. thermophilum were 2.96 mg mL -1 and 0.25 µmol min -1 mg -1 , respectively. These findings in this study have great implications for the future applications of xylanases.

Action of fungal laccases on lignin model compounds in organic solvents

Abstract The stability and reactivity of five different thermostable fungal laccases from the species Trametes hirsuta , Melanocarpus albomyces , Thielavia arenaria (two laccases) and Chaetomium thermophilum were investigated in the presence of organic solvents. Oxidations of small organic phenolic compounds, matairesinol and 7-hydroxymatairesinol lignans, as well as synthetic lignin dehydrogenation polymer DHP in aqueous solutions of ethanol and propylene glycol solvents were investigated using analysis of oxidation rates, high performance liquid chromatography and size-exclusion chromatography. The laccases showed variability in their solvent tolerance. The redox potential of the laccases appeared not to be the main factor determining the efficiency of the polymerization reactions of complex phenolic model compounds in aqueous organic solutions. Nuclear magnetic resonance spectroscopic analysis of laccase treated DHP in 50% propylene glycol indicated that the formation of new biphenylic 5–5′ structures was favored in laccase-catalyzed radical coupling reactions over the other possible reactions through the phenolic groups forming new 5 O 4 ether bonds. The polymerization reactions took place even at high concentrations of solvents, which already inhibited the enzyme activity, encouraging enzymatic upgrading of lignin in organic solvents to be studied further. Thus, it was confirmed that thermostable laccases are potential enzymes for various industrial applications where organic solvents are required for the reaction systems.

Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum

Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4-5days after methanol induction compared to 6days for the wild-type. The optimum reaction temperature (60°C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50°C and pH 4). Kept at 80°C for 1h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.

Production, Partial Purification and Some Properties of α-L-Arabinofuranosidase from Chaetomium thermophile and Talaromyces thermophilus

Production, Partial Purification and Some Properties of α-L-Arabinofuranosidase from Chaetomium thermophile and Talaromyces thermophilus

Analysis of the yeast nucleoporin Nup188 reveals a conserved S-like structure with similarity to karyopherins

Nuclear pore complexes (NPCs) embedded in the double nuclear membrane mediate the entire nucleocytoplasmic transport between the nucleus and cytoplasm. Each NPC is composed of about 30 different proteins (nucleoporins or Nups), which exist in multiple (8, 16 or 32) copies within the NPC scaffold. Recently, we have identified and characterized the large structural Nups, Nup188 and Nup192, from the thermophilic eukaryote Chaetomium thermophilum, which exhibited superior properties for biochemical and structural studies, when compared to their mesophilic orthologs. Here, we study the large structural Nups from the model organism yeast Saccharomyces cerevisiae. Our data show that yeast Nup188 like its thermophilic orthologue ctNup188 exhibits a twisted S-like structure, which flexibly binds the linker nucleoporin Nic96 via a short conserved α-helix motif. Using bioinformatic methods, we have generated a pseudo-atomic structural model of Nup188 and its related Nup192, which further strengthens the view that the large α-solenoid structural Nups are related to karyopherins.