Defective recognition of a nonclassical nuclear localization signal in neurodevelopmental disorders.

In this issue of Structure, Gonzalez et al. present the cryo-EM structure of Karyopherin-ß2 bound to the proline-tyrosine nuclear localization signal (PY-NLS) of heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2). The structure advances our understanding of not only the diversity of PY-NLSs but also the pathogenic mechanisms arising from HNRNPH2 variants.

Toxoplasma Gondii Importin α Shows Weak Auto-Inhibition.

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin ß-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

A new Karyopherin-ß2 binding PY-NLS epitope of HNRNPH2 linked to neurodevelopmental disorders.

The HNRNPH2 proline-tyrosine nuclear localization signal (PY-NLS) is mutated in HNRNPH2-related X-linked neurodevelopmental disorder, causing the normally nuclear HNRNPH2 to accumulate in the cytoplasm. We solved the cryoelectron microscopy (cryo-EM) structure of Karyopherin-ß2/Transportin-1 bound to the HNRNPH2 PY-NLS to understand importin-NLS recognition and disruption in disease. HNRNPH2 206RPGPY210 is a typical R-X2-4-P-Y motif comprising PY-NLS epitopes 2 and 3, followed by an additional Karyopherin-ß2-binding epitope, we term epitope 4, at residues 211DRP213; no density is present for PY-NLS epitope 1. Disease variant mutations at epitopes 2-4 impair Karyopherin-ß2 binding and cause aberrant cytoplasmic accumulation in cells, emphasizing the role of nuclear import defect in disease. Sequence/structure analysis suggests that strong PY-NLS epitopes 4 are rare and thus far limited to close paralogs of HNRNPH2, HNRNPH1, and HNRNPF. Epitope 4-binidng hotspot Karyopherin-ß2 W373 corresponds to close paralog Karyopherin-ß2b/Transportin-2 W370, a pathological variant site in neurodevelopmental abnormalities, suggesting that Karyopherin-ß2b/Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities.

A minimal construct of nuclear-import receptor Karyopherin-ß2 defines the regions critical for chaperone and disaggregation activity.

Karyopherin-ß2 (Kapß2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals of diverse cytoplasmic cargo for transport to the nucleus. Kapß2 cargo includes several disease-linked RNA-binding proteins with prion-like domains, such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RNA-binding proteins with prion-like domains are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Remarkably, Kapß2 prevents and reverses aberrant phase transitions of these cargoes, which is cytoprotective. However, the molecular determinants of Kapß2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapß2 is a super-helical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapß2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8 to 20 of Kapß2 recapitulate all salient features of Kapß2 activity. By contrast, Kapß2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapß2 construct for delivery in adeno-associated viruses as a potential therapeutic for amyotrophic lateral sclerosis/frontotemporal dementia, multisystem proteinopathy, and related disorders.

Biochemical propensity mapping for structural and functional anatomy of importin α IBB domain.

Importin α has been described as a nuclear protein transport receptor that enables proteins synthesized in the cytoplasm to translocate into the nucleus. Besides its function in nuclear transport, an increasing number of studies have examined its non-nuclear transport functions. In both nuclear transport and non-nuclear transport, a functional domain called the IBB domain (importin ß binding domain) plays a key role in regulating importin α behavior, and is a common interacting domain for multiple binding partners. However, it is not yet fully understood how the IBB domain interacts with multiple binding partners, which leads to the switching of importin α function. In this study, we have distinguished the location and propensities of amino acids important for each function of the importin α IBB domain by mapping the biochemical/physicochemical propensities of evolutionarily conserved amino acids of the IBB domain onto the structure associated with each function. We found important residues that are universally conserved for IBB functions across species and family members, in addition to those previously known, as well as residues that are presumed to be responsible for the differences in complex-forming ability among family members and for functional switching.

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers.

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

Comparative study of the interaction of ivermectin with proteins of interest associated with SARS-CoV-2: A computational and biophysical approach.

The SARS-CoV-2 pandemic has accelerated the study of existing drugs. The mixture of homologs called ivermectin (avermectin-B1a [HB1a] + avermectin-B1b [HB1b]) has shown antiviral activity against SARS-CoV-2 in vitro. However, there are few reports on the behavior of each homolog. We investigated the interaction of each homolog with promising targets of interest associated with SARS-CoV-2 infection from a biophysical and computational-chemistry perspective using docking and molecular dynamics. We observed a differential behavior for each homolog, with an affinity of HB1b for viral structures, and of HB1a for host structures considered. The induced disturbances were differential and influenced by the hydrophobicity of each homolog and of the binding pockets. We present the first comparative analysis of the potential theoretical inhibitory effect of both avermectins on biomolecules associated with COVID-19, and suggest that ivermectin through its homologs, has a multiobjective behavior.

Karyopherin-ßs play a key role as a phase separation regulator.

Recent studies have revealed that cells utilize liquid-liquid phase separation (LLPS) as a mechanism in assembly of membrane-less organelles, such as RNP granules. The nucleus is a well-known membrane-bound organelle surrounded by the nuclear envelope; the nuclear pore complex on the nuclear envelope likely applies LLPS in the central channel to facilitate selective biological macromolecule exchange. Karyopherin-ß family proteins exclusively pass through the central channel with cargos by dissolving the phase separated hydrogel formed by the phenylalanine-glycine (FG) repeats-containing nucleoporins. Karyopherin-ßs also exhibit dissolution activity for the phase separation of cargo proteins. Many cargos, including RNA-binding proteins containing intrinsically disordered regions (IDRs), undergo phase separation; however, aberrant phase separation is linked to fatal neurodegenerative diseases. Multiple weak interactions between karyopherin-ßs and phase separation-prone proteins, such as FG repeats-containing nucleoporins or IDR-containing karyopherin-ß cargos, are likely to be important for passing through the nuclear pore complex and maintaining the soluble state of cargo, respectively. In this review, we discuss how karyopherin-ßs regulate phase separation to function.

Pharmacoinformatics based elucidation and designing of potential inhibitors against Plasmodium falciparum to target importin α/ß mediated nuclear importation.

Plasmodium falciparum, the prime causative agent of malaria, is responsible for 4, 05,000 deaths per year and fatality rates are higher among the children aged below 5 years. The emerging distribution of the multi-drug resistant P. falciparum becomes a worldwide concern, so the identification of unique targets and novel inhibitors is a prime need now. In the present study, we have employed pharmacoinformatics approaches to analyze 265 lead-like compounds from PubChem databases for virtual screening. Thereafter, 15 lead-like compounds were docked within the active side pocket of importin alpha. Comparative ligand properties and absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile were also assessed. Finally, a novel inhibitor was designed and assessed computationally for its efficacy. From the comparative analysis we have found that our screened compounds possess better results than the existing lead ivermectin; having the highest binding energy of -15.6 kcal/mol, whereas ivermectin has -12.4 kcal/mol. The novel lead compound possessed more fascinating output without deviating any of the rules of Lipinski. It also possessed higher bioavailability and the drug-likeness score of 0.55 and 0.71, respectively compared to ivermectin. Furthermore, the binding study was confirmed by molecular dynamics simulation over 25 ns by evaluating the stability of the complex. Finally, all the screened compounds and the novel compound showed promising ADMET properties likewise. To end, we hope that our proposed screened compounds, as well as the novel compound, might give some advances to treat malaria efficiently in vitro and in vivo.

Karyopherins and condensates.

Several aggregation-prone RNA-binding proteins, including FUS, EWS, TAF15, hnRNP A1, hnRNP A2, and TDP-43, are mutated in neurodegenerative diseases. The nuclear-cytoplasmic distribution of these proteins is controlled by proteins in the karyopherin family of nuclear transport factors (Kaps). Recent studies have shown that Kaps not only transport these proteins but also inhibit their self-association/aggregation, acting as molecular chaperones. This chaperone activity is impaired for disease-causing mutants of the RNA-binding proteins. Here, we review physical data on the mechanisms of self-association of several disease-associated RNA-binding proteins, through liquid-liquid phase separation and amyloid fiber formation. In each case, we relate these data to biophysical, biochemical, and cell biological data on the inhibition of self-association by Kaps. Our analyses suggest that Kaps may be effective chaperones because they contain large surfaces with diverse physical properties that enable them to engage multiple different regions of their cargo proteins, blocking self-association.

Nonclassical nuclear localization signals mediate nuclear import of CIRBP.

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

Nup358 and Transportin 1 Cooperate in Adenoviral Genome Import.

Nuclear import of viral genomes is an important step during the life cycle of adenoviruses (AdV), requiring soluble cellular factors as well as proteins of the nuclear pore complex (NPC). We addressed the role of the cytoplasmic nucleoporin Nup358 during adenoviral genome delivery by performing depletion/reconstitution experiments and time-resolved quantification of adenoviral genome import. Nup358-depleted cells displayed reduced efficiencies of nuclear import of adenoviral genomes, and the nuclear import receptor transportin 1 became rate limiting under these conditions. Furthermore, we identified a minimal N-terminal region of Nup358 that was sufficient to compensate for the import defect. Our data support a model where Nup358 functions as an assembly platform that promotes the formation of transport complexes, allowing AdV to exploit a physiological protein import pathway for accelerated transport of its DNA.IMPORTANCE Nuclear import of viral genomes is an essential step to initiate productive infection for several nuclear replicating DNA viruses. On the other hand, DNA is not a physiological nuclear import substrate; consequently, viruses have to exploit existing physiological transport routes. Here, we show that adenoviruses use the nucleoporin Nup358 to increase the efficiency of adenoviral genome import. In its absence, genome import efficiency is reduced and the transport receptor transportin 1 becomes rate limiting. We show that the N-terminal half of Nup358 is sufficient to drive genome import and identify a transportin 1 binding region. In our model, adenovirus genome import exploits an existing protein import pathway and Nup358 serves as an assembly platform for transport complexes.

X-ray Structure of the Human Karyopherin RanBP5, an Essential Factor for Influenza Polymerase Nuclear Trafficking.

Here, we describe the crystal structures of two distinct isoforms of ligand-free human karyopherin RanBP5 and investigate its global propensity to interact with influenza A virus polymerase. Our results confirm the general architecture and mechanism of the IMB3 karyopherin-ß subfamily whilst also highlighting differences with the yeast orthologue Kap121p. Moreover, our results provide insight into the structural flexibility of ß-importins in the unbound state. Based on docking of a nuclear localisation sequence, point mutations were designed, which suppress influenza PA-PB1 subcomplex binding to RanBP5 in a binary protein complementation assay.

Genome-Wide Identification, Expression Profile and Evolution Analysis of Karyopherin ß Gene Family in Solanum tuberosum Group Phureja DM1-3 Reveals Its Roles in Abiotic Stresses.

In eukaryotic cells, nucleocytoplasmic trafficking of macromolecules is largely mediated by Karyopherin ß/Importin (KPNß or Impß) nuclear transport factors, and they import and export cargo proteins or RNAs via the nuclear pores across the nuclear envelope, consequently effecting the cellular signal cascades in response to pathogen attack and environmental cues. Although achievements on understanding the roles of several KPNßs have been obtained from model plant Arabidopsis thaliana, comprehensive analysis of potato KPNß gene family is yet to be elucidated. In our genome-wide identifications, a total of 13 StKPNß (Solanum tuberosum KPNß) genes were found in the genome of the doubled monoploid S. tuberosum Group Phureja DM1-3. Sequence alignment and conserved domain analysis suggested the presence of importin-ß N-terminal domain (IBN_N, PF08310) or Exporin1-like domain (XpoI, PF08389) at N-terminus and HEAT motif at the C-terminal portion in most StKPNßs. Phylogenetic analysis indicated that members of StKPNß could be classified into 16 subgroups in accordance with their homology to human KPNßs, which was also supported by exon-intron structure, consensus motifs, and domain compositions. RNA-Seq analysis and quantitative real-time PCR experiments revealed that, except StKPNß3d and StKPNß4, almost all StKPNßs were ubiquitously expressed in all tissues analyzed, whereas transcriptional levels of several StKPNßs were increased upon biotic/abiotic stress or phytohormone treatments, reflecting their potential roles in plant growth, development or stress responses. Furthermore, we demonstrated that silencing of StKPNß3a, a SA- and H2O2-inducible KPNß genes led to increased susceptibility to environmental challenges, implying its crucial roles in plant adaption to abiotic stresses. Overall, our results provide molecular insights into StKPNß gene family, which will serve as a strong foundation for further functional characterization and will facilitate potato breeding programs.

Importin-ß/karyopherin-ß1 modulates mitotic microtubule function and taxane sensitivity in cancer cells via its nucleoporin-binding region.

The nuclear transport receptor importin-ß/karyopherin-ß1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-ß regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-ß's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-ß form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-ß affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-ß interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-ß, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-ß's nucleoporin-binding residues. Concomitantly, importin-ß overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-ß function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs.

Transportin-1 binds to the HIV-1 capsid via a nuclear localization signal and triggers uncoating.

The initial steps of HIV replication in host cells prime the virus for passage through the nuclear pore and drive the establishment of a productive and irreparable infection1,2. The timely release of the viral genome from the capsid-referred to as uncoating-is emerging as a critical parameter for nuclear import, but the triggers and mechanisms that orchestrate these steps are unknown. Here, we identify ß-karyopherin Transportin-1 (TRN-1) as a cellular co-factor of HIV-1 infection, which binds to incoming capsids, triggers their uncoating and promotes viral nuclear import. Depletion of TRN-1, which we characterized by mass spectrometry, significantly reduced the early steps of HIV-1 infection in target cells, including primary CD4+ T cells. TRN-1 bound directly to capsid nanotubes and induced dramatic structural damage, indicating that TRN-1 is necessary and sufficient for uncoating in vitro. Glycine 89 on the capsid protein, which is positioned within a nuclear localization signal in the cyclophilin A-binding loop, is critical for engaging the hydrophobic pocket of TRN-1 at position W730. In addition, TRN-1 promotes the efficient nuclear import of both viral DNA and capsid protein. Our study suggests that TRN-1 mediates the timely release of the HIV-1 genome from the capsid protein shell and efficient viral nuclear import.

Characterization of the Importin-ß binding domain in nuclear import receptor KPNA7.

The KPNA family of mammalian nuclear import receptors are encoded by seven genes that generate isoforms with 42-86% identity. KPNA isoforms have the same protein architecture and share the functional property of nuclear localization signal (NLS) recognition, however, the tissue and developmental expression patterns of these receptors raise the question of whether subtle differences in KPNA isoforms might be important in specific biological contexts. Here, we show that KPNA7, an isoform with expression mostly limited to early development, can bind Importin-ß (Imp-ß) in the absence of NLS cargo. This result contrasts with Imp-ß interactions with other KPNA family members, where affinity is regulated by NLS cargo as part of a cooperative binding mechanism. The Imp-ß binding (IBB) domain, which is highly conserved in all KPNA family members, generally serves to occlude the NLS binding groove and maintain the receptor in an auto-inhibited 'closed' state prior to NLS contact. Cooperative binding of NLS cargo and Imp-ß to KPNA results in an 'open'state. Characterization of KPNA2-KPNA7 chimeric proteins suggests that features of both the IBB domain and the core structure of the receptor contribute to the extent of IBB domain accessibility for Imp-ß binding, which likely reflects an 'open' state. We also provide evidence that KPNA7 maintains an open-state in the nucleus. We speculate that KPNA7 could function within the nucleus by interacting with NLS-containing proteins.

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.

Differential role for phosphorylation in alternative polyadenylation function versus nuclear import of SR-like protein CPSF6.

Cleavage factor I mammalian (CFIm) complex, composed of cleavage and polyadenylation specificity factor 5 (CPSF5) and serine/arginine-like protein CPSF6, regulates alternative polyadenylation (APA). Loss of CFIm function results in proximal polyadenylation site usage, shortening mRNA 3' untranslated regions (UTRs). Although CPSF6 plays additional roles in human disease, its nuclear translocation mechanism remains unresolved. Two ß-karyopherins, transportin (TNPO) 1 and TNPO3, can bind CPSF6 in vitro, and we demonstrate here that while the TNPO1 binding site is dispensable for CPSF6 nuclear import, the arginine/serine (RS)-like domain (RSLD) that mediates TNPO3 binding is critical. The crystal structure of the RSLD-TNPO3 complex revealed potential CPSF6 interaction residues, which were confirmed to mediate TNPO3 binding and CPSF6 nuclear import. Both binding and nuclear import were independent of RSLD phosphorylation, though a hyperphosphorylated mimetic mutant failed to bind TNPO3 and mislocalized to the cell cytoplasm. Although hypophosphorylated CPSF6 largely supported normal polyadenylation site usage, a significant number of mRNAs harbored unnaturally extended 3' UTRs, similar to what is observed when other APA regulators, such as CFIIm component proteins, are depleted. Our results clarify the mechanism of CPSF6 nuclear import and highlight differential roles for RSLD phosphorylation in nuclear translocation versus regulation of APA.

Mechanism-Dependent Modulation of Ultrafast Interfacial Water Dynamics in Intrinsically Disordered Protein Complexes.

The recognition of intrinsically disordered proteins (IDPs) is highly dependent on dynamics owing to the lack of structure. Here we studied the interplay between dynamics and molecular recognition in IDPs with a combination of time-resolving tools on timescales ranging from femtoseconds to nanoseconds. We interrogated conformational dynamics and surface water dynamics and its attenuation upon partner binding using two IDPs, IBB and Nup153FG, both of central relevance to the nucleocytoplasmic transport machinery. These proteins bind the same nuclear transport receptor (Importinß) with drastically different binding mechanisms, coupled folding-binding and fuzzy complex formation, respectively. Solvent fluctuations in the dynamic interface of the Nup153FG-Importinß fuzzy complex were largely unperturbed and slightly accelerated relative to the unbound state. In the IBB-Importinß complex, on the other hand, substantial relative slowdown of water dynamics was seen in a more rigid interface. These results show a correlation between interfacial water dynamics and the plasticity of IDP complexes, implicating functional relevance for such differential modulation in cellular processes, including nuclear transport.