Biology subjects

Chook, Y. M.

Publications and source records attributed to Chook, Y. M..

12 recordsLinked to original sources

bioRxiv2026

CliPepPI: Scalable prediction of domain-peptide specificityusing contrastive learning

Domain-peptide interactions mediate a significant fraction of cellular protein networks, yet accurately predicting their specificity remains challenging. Peptide motifs typically have short, fuzzy sequence profiles, and their interactions are often weak and transient, limiting the size, coverage, and quality of experimentally validated domain-peptide datasets. Since true non-binders are rarely known, constructing negative examples often introduces bias. While structure-based prediction methods can achieve high accuracy, they are computationally demanding and difficult to scale to the proteome level. We introduce CLIPepPI, a dual-encoder model that leverages contrastive learning to embed domains and peptides into a shared space directly from sequence. Both encoders are initialized from a protein language model (ESM-C) and fine-tuned using lightweight LoRA adapters, enabling parameter-efficient training on positive pairs alone. To overcome data scarcity, we augment [~]3K protein-peptide complexes from PPI3D with [~]150K domain-peptide pairs derived from protein-protein interfaces. CLIPepPI further injects structural information by marking interface residues in the domain sequence, thus guiding the encoders toward binding regions and linking sequence-level learning with structural context. Competitive performance is achieved across three independent benchmarks: domain-peptide complexes from PPI3D, large-scale phage-library data from ProP-PD, and a curated dataset of nuclear export signal (NES) sequences. We demonstrate scalability and generalization through two applications: (i) proteome-wide NES scanning, and (ii) variant-effect prediction, where score changes in domain-peptide interactions between wild-type and mutant sequences discriminate pathogenic from benign variants. Together, CLIPepPI offers a scalable, structure-informed model for predicting domain-peptide specificity and generating meaningful embeddings suited for large-scale proteomic analyses. CLIPepPI is available at: https://bio3d.cs.huji.ac.il/webserver/clipeppi/.

bioinformatics↗

bioRxiv2026

Importins recognize the winged-helix fold of ETS transcription factors to mediate nuclear import

Protein trafficking between the cytoplasm and the nucleus is a fundamental process in eukaryotic cell biology. While linear nuclear localization signals (NLSs) are well-characterized, many nuclear proteins lack a predictable NLS. Here, we identify the ETS domain, a DNA-binding winged-helix fold, from ETS family transcription factors as a structure-encoded NLS. We show that ETS domains mediate nuclear import through direct recognition by multiple nuclear transport receptors, including IPO9. Cryo-electron microscopy analysis of the EHF:IPO9 complex reveals that the IPO9 wraps around the ETS domain and engages structural features throughout the winged-helix fold. Biochemical studies demonstrate that the ETS domain DNA-binding helix is critical for importin recognition and for NLS activity in mammalian cells. Comparison of IPO9 bound to EHF and the histone H2A:H2B dimer reveals distinct interaction hotspots, illustrating how IPO9 employs unique combinatorial binding surfaces to accommodate structurally diverse cargos. These findings define a new class of globular NLSs and highlight the adaptability of importins in recognizing distinct protein folds. Significance StatementNuclear import is essential for transcription factor function. However, many nuclear proteins lack recognizable nuclear localization signals (NLSs), leaving their trafficking mechanisms unresolved. Here, we identify the winged-helix DNA-binding domain of ETS transcription factors as a structure-encoded NLS shared across the ETS family of proteins. We show that multiple importins directly interact with this globular domain and define the molecular basis for cargo recognition by determining the cryo-EM structure of an importin bound to an ETS family protein. These studies establish a new class of globular NLSs and shed light on how individual importins can recognize diverse protein folds. We also provide mechanistic insight into nuclear trafficking defects that are caused by disease-linked ETS transcription factor mutations.

biochemistry↗

bioRxiv2024

Setdb1 and Atf7IP form a hetero-trimeric complex that blocks Setdb1 nuclear export

AbstractHistone H3K9 methylation (H3K9me) by Setdb1 silences retrotransposons (rTE) by sequestering them in constitutive heterochromatin. Atf7IP is a constitutive binding partner of Setdb1 and is responsible for Setdb1 nuclear localization, activation and chromatin recruitment. However, structural details of the Setdb1/Atf7IP interaction have not been evaluated. We used Alphafold2 predictions and biochemical reconstitutions to show that one copy of Setdb1 and two copies of Atf7IP form a hetero-trimeric complex in vitro and in cells. We also find that Atf7IP self-associates, forming multimeric complexes that are resolved upon Setdb1 binding. Setdb1 binds to Atf7IP through coiled coil interactions that include both Setdb1 nuclear export signals (NES). Atf7IP directly competes with CRM1 to bind the Setdb1 NES motifs, explaining how Atf7IP prevents CRM1-mediated nuclear export of Setdb1. Setdb1 also forms hetero-trimeric complexes with the Atf7IP paralog Atf7IP2 and we show that Setdb1 can form mixed heterotrimers comprising one copy of each Setdb1, Atf7IP and Atf7IP2. Atf7IP and Atf7IP2 are co-expressed in many tissues suggesting that heterotrimers with different compositions of Atf7IP and Atf7IP2 may differentially regulate H3K9me by fine-tuning Setdb1 localization and activity.

biochemistry↗

bioRxiv2024

Allosteric degraders induce CRL5ASB8 mediated degradation of XPO1

SummaryThe nuclear export receptor exportin 1 (XPO1/CRM1) is often overexpressed in cancer cells, leading to the mislocalization of numerous cancer-related protein cargoes1,2. Selinexor, a covalent XPO1 inhibitor, and other Selective Inhibitor of Nuclear Export (SINEs) restore proper nuclear localization by blocking XPO1-cargo binding2-7. SINEs also induce XPO1 degradation via the Cullin-RING E3 ubiquitin ligase (CRL) substrate receptor ASB87. Here we elucidate the mechanism underlying the high-affinity engagement of CRL5ASB8 with SINE-conjugated XPO1. Cryogenic electron microscopy (cryoEM) structures reveal that ASB8 binds to a cryptic site on XPO1, which becomes accessible only upon SINE conjugation. While molecular glue degraders typically interact with both CRL and the substrate8-10, SINEs bind to XPO1 without requiring interaction with ASB8 for efficient XPO1 degradation. Instead, an allosteric mechanism facilitates high affinity XPO1-ASB8 interaction, leading to XPO1 ubiquitination and degradation. ASB8-mediated degradation is also observed upon treatment of the endogenous itaconate derivate 4-octyl itaconate, which suggests a native mechanism that is inadvertently exploited by synthesized XPO1 inhibitors. This allosteric XPO1 degradation mechanism of SINE compounds expands the known modes of targeted protein degradation beyond the well-characterized molecular glue degraders and proteolysis targeting chimeras of CRL4.

cancer biology↗

bioRxiv2024

Phosphate-dependent nuclear export via a novel NES class recognized by exportin Msn5

Gene expression in response to environmental stimuli is dependent on nuclear localization of key signaling components, which can be tightly regulated by phosphorylation. This is exemplified by the phosphate-sensing transcription factor Pho4, which requires phosphorylation for nuclear export by the yeast exportin Msn5. Unlike the traditional hydrophobic nuclear export signal (NES) utilized by the Exportin-1/XPO1 system, cryogenic-electron microscopy structures reveal that Pho4 presents a novel, phosphorylated 35-residue NES that interacts with the concave surface of Msn5 through two Pho4 phospho-serines that align with two Msn5 basic patches, unveiling a previously unknown mechanism of phosphate-specific recognition. Furthermore, the discovery that unliganded Msn5 is autoinhibited explains the positive cooperativity of Pho4/Ran-binding and proposes a mechanism for Pho4s release in the cytoplasm. These findings advance our understanding of the diversity of signals that drive nuclear export and how cargo phosphorylation is crucial in regulating nuclear transport and controlling cellular signaling pathways.

biophysics↗

bioRxiv2024

Enigmatic missense mutations can cause disease via creation of de novo nuclear export signals

Disease-causing missense mutations that occur within structurally and functionally unannotated protein regions can guide researchers to new mechanisms of protein regulation and dysfunction. Here, we report that the thrombocytopenia-, myelodysplastic syndromes-, and leukemia-associated P214L mutation in the transcriptional regulator ETV6 creates an XPO1-dependent nuclear export signal to cause protein mislocalization. Strategies to disrupt XPO1 activity fully restore ETV6 P214L protein nuclear localization and transcription regulation activity. Mechanistic insight inspired the design of a humanized ETV6 mice, which we employ to demonstrate that the germline P214L mutation is sufficient to elicit severe defects in thrombopoiesis and hematopoietic stem cell maintenance. Beyond ETV6, we employed computational methods to uncover rare disease-associated missense mutations in unrelated proteins that create a nuclear export signal to disrupt protein function. Thus, missense mutations that operate through this mechanism should be predictable and may suggest rational therapeutic strategies for associated diseases.

biochemistry↗

bioRxiv2023

Structural mechanism of co-import of H2A-H2B and its chaperone Nap1 by nuclear import receptor Kap114

Core histones are synthesized and processed in the cytoplasm before transport into the nucleus for assembly into nucleosomes; however, they must also be chaperoned as free histones are toxic. The importin Kap114 binds and transports histone H2A-H2B into the yeast nucleus, where RanGTP facilitates H2A-H2B release. Kap114 and H2A-H2B also bind the Nap1 histone chaperone, which is found in both the cytoplasm and the nucleus, but how Nap1 and Kap114 cooperate in H2A-H2B processing and nucleosome assembly has been unclear. To understand these mechanisms, we used biochemical and structural analyses to reveal how Nap1, Kap114, H2A-H2B and RanGTP interact. We show that Kap114, H2A-H2B and a Nap1 dimer (Nap12) assemble into a 1:1:1 ternary complex. Cryogenic electron microscopy revealed two distinct Kap114/Nap12/H2A-H2B structures: one of H2A-H2B sandwiched between Nap12 and Kap114, and another in which Nap12 bound to the Kap114*H2A-H2B complex without contacting H2A-H2B. Another Nap12*H2A-H2B*Kap114*RanGTP structure reveals the nuclear complex. Mutagenesis revealed shared critical interfaces in all three structures. Consistent with structural findings, DNA competition experiments demonstrated that Kap114 and Nap12 together chaperone H2A-H2B better than either protein alone. When RanGTP is present, Kap114s chaperoning activity diminishes. However, the presence of Nap12 within the Nap12*H2A-H2B*Kap114*RanGTP quaternary complex restores its ability to chaperone H2A-H2B. This complex effectively deposits H2A-H2B into nucleosomes. Together, these findings suggest that Kap114 and Nap12 provide a sheltered path from cytoplasm to nucleus, facilitating the transfer of H2A-H2B from Kap114 to Nap12, ultimately directing its specific deposition into nucleosomes. Significance StatementFree histones are toxic and must be sequestered by other macromolecules in the cell. Nuclear import receptor Kap114 imports H2A-H2B into the nucleus while also chaperoning it. The histone chaperone Nap1 also chaperones H2A-H2B, but it is unclear how Nap1 and Kap114 cooperate to process H2A-H2B. We present biochemical and structural results that explain how Kap114, Nap1 and H2A-H2B assemble in the absence and presence of RanGTP, how Nap1 and Kap114 co-chaperone H2A-H2B, and how RanGTP and Nap1 coordinate the transfer of H2A-H2B from Kap114 to assembling nucleosomes in the nucleus.

biophysics↗

bioRxiv2023

Molecular basis of RanGTP-activated nucleosome assembly with Histones H2A-H2B bound to Importin-9

Padavannil et al. 2019 show that Importin-9 (Imp9) transports Histones H2A-H2B from the cytoplasm to the nucleus using a non-canonical mechanism whereby binding of a GTP-bound Ran GTPase (RanGTP) fails to evict the H2A-H2B cargo. Instead, a stable complex forms, comprised of equimolar RanGTP, Imp9, and H2A-H2B. Unlike the binary Imp9*H2A-H2B complex, this RanGTP*Imp9*H2A-H2B ternary complex can release H2A-H2B to an assembling nucleosome. Here, we define the molecular basis for this RanGTP-activated nucleosome assembly by Imp9. We use hydrogen-deuterium exchange coupled with mass spectrometry and compare the dynamics and interfaces of the RanGTP*Imp9*H2A-H2B ternary complex to those in the Imp9*H2A-H2B or Imp9*RanGTP binary complexes. Our data are consistent with the Imp9*H2A-H2B structure by Padavannil et al. 2019 showing that Imp9 HEAT repeats 4-5 and 18-19 contact H2A-H2B, as well as many homologous importin*RanGTP structures showing that importin HEAT repeats 1 and 3, and the h8 loop, contact RanGTP. We show that Imp9 stabilizes H2A-H2B beyond the direct binding site, similar to other histone chaperones. Importantly, we reveal that binding of RanGTP releases H2A-H2B interaction at Imp9 HEAT repeats 4-5, but not 18-19. This exposes DNA- and histone-binding surfaces of H2A-H2B, thereby facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. This may ensure that H2A-H2B is only released in high RanGTP concentrations near chromatin. We delineate the molecular link between the nuclear import of H2A-H2B and its deposition into chromatin by Imp9. SignificanceImp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP alone is insufficient to release H2A-H2B. The resulting stable RanGTP*Imp9*H2A-H2B complex gains nucleosome assembly activity as H2A-H2B can be deposited onto an assembling nucleosome. We show that H2A-H2B is allosterically stabilized via interactions with both N- and C-terminal portions of Imp9, reinforcing its chaperone-like behavior. RanGTP binding causes H2A-H2B release from the N-terminal portion of Imp9 only. The newly-exposed H2A-H2B surfaces can interact with DNA or H3-H4 in nucleosome assembly. Imp9 thus plays a multi-faceted role in histone import, storage, and deposition regulated by RanGTP, controlling histone supply in the nucleus and to chromatin.

biophysics↗

bioRxiv2023

A new Karyopherin-β2 binding PY-NLS epitope of HNRNPH2 is linked to neurodevelopmental disorders

The normally nuclear HNRNPH2 is mutated in HNRNPH2-related X-linked neurodevelopmental disorder causing the protein to accumulate in the cytoplasm. Interactions of HNRNPH2 with its importin Karyopherin-{beta}2 (Transportin-1) had not been studied. We present a structure that shows Karyopherin-{beta}2 binding HNRNPH2 residues 204-215, a proline-tyrosine nuclear localization signal or PY-NLS that contains a typical R-X2-4-P-Y motif, 206RPGPY210, followed a new Karyopherin-{beta}2 binding epitope at 211DRP213 that make many interactions with Karyopherin-{beta}2 W373. Mutations at each of these sites decrease Karyopherin-{beta}2 binding affinities by 70-100 fold, explaining aberrant accumulation in cells and emphasizing the role of nuclear import defects in the disease. Sequence/structure analysis suggests that the new epitope C-terminal of the PY-motif, which binds Karyopherin-{beta}2 W373, is rare and thus far limited to close paralogs HNRNPH2, HNRNPH1 and HNRNPF. Karyopherin-{beta}2 W373, a HNRNPH2-binding hotspot, corresponds to W370 of close paralog Transportin-2, a site of pathological variants in patients with neurodevelopmental abnormalities, suggesting that Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities. SummaryHNRNPH2 variants in HNRNPH2-related X-linked neurodevelopmental disorder aberrantly accumulate in the cytoplasm. A structure of Karyopherin-{beta}2*HNRNPH2 explains nuclear import defects of the variants, reveals a new NLS epitope that suggests mechanistic changes in pathological variants of Karyopherin-{beta}2 paralog Transportin-2.

biophysics↗

bioRxiv2022

Mechanism of RanGTP priming the release of H2A-H2B from Kap114 and Importin-9

Previously we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9*H2A-H2B complex and formation of the ternary RanGTP*Importin-9*H2A-H2B complex facilitates H2A-H2B release to the assembling nucleosome. It was unclear how RanGTP and the cargo H2A-H2B can bind simultaneously to an importin, and how interactions of the three components position H2A-H2B for nucleosome assembly. Here we show cryo-EM structures of Importin-9*RanGTP and of its yeast homolog Kap114, including Kap114*RanGTP, Kap114*H2A-H2B, and RanGTP*Kap114*H2A-H2B to explain how the conserved Kap114 binds H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. In the ternary complex, RanGTP binds to the N-terminal repeats of Kap114 in the same manner as in the Kap114/Importin-9*RanGTP complex, and H2A-H2B binds via its acidic patch to the Kap114 C-terminal repeats much like in the Kap114/Importin-9*H2A-H2B complex. Ran binds to a different conformation of Kap114 in the ternary RanGTP*Kap114*H2A-H2B complex. Here, Kap114 no longer contacts the H2A-H2B surface proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome. Significance StatementHistones and their chaperone networks are typically conserved in eukaryotes. The yeast importin Kap114 and its human homolog Importin-9 share low sequence identity, but both are primary nuclear import receptors for the core histone heterodimer H2A-H2B. Cryo-EM structures of Kap114*H2A-H2B, Kap114*RanGTP and Importin-9*RanGTP complexes show homologous structure and function for Kap114 and Importin-9. In the nucleus, RanGTP binding to Kap114/Imp9*H2A-H2B does not release H2A-H2B, but RanGTP binds to form an atypical ternary complex. Structure of the ternary RanGTP*Kap114*H2A-H2B complex explains how the GTPase and cargo bind simultaneously to Kap114 and how the presence of Ran in the complex primes H2A-H2B transfer to assembling nucleosomes.

biochemistry↗

bioRxiv2022

Structure of Importin-4 bound to the H3-H4·ASF1 histone·histone chaperone complex

Importin-4 is the primary nuclear import receptor of core histones H3 and H4. Importin-4 binds the H3-H4 dimer and histone-chaperone ASF1 prior to nuclear import, but available structures of Importin-4{middle dot}histone tail complexes do not explain how Importin-4 recognizes the biologically relevant heterotrimeric H3-H4{middle dot}ASF1 cargo. Our 3.5 [A] Importin-4{middle dot}H3-H4{middle dot}ASF1 cryo-electron microscopy structure revealed interactions with H3-H4{middle dot}ASF1 different those suggested by previous Importin-H3 tail peptide structures. The N-terminal half of Importin-4 clamps the globular histone domain and the H3 N helix while its C-terminal half binds the H3 N-terminal tail weakly, with negligible tail contribution to binding energy; ASF1 binds H3-H4 without contacting Importin-4. Together, ASF1 and Importin-4 shield nucleosomal interfaces of H3-H4 to chaperone and import it into the nucleus, where Importin-4 undergoes large conformational changes as RanGTP binds to release H3-H4{middle dot}ASF1. This work explains the mechanisms of nuclear import of full-length H3-H4.

molecular biology↗

bioRxiv2022

A murine model of hnRNPH2-related neurodevelopmental disorder recapitulates clinical features of human disease and reveals a mechanism for genetic compensation of HNRNPH2

Mutations in HNRNPH2 cause an X-linked neurodevelopmental disorder with a phenotypic spectrum that includes developmental delay, intellectual disability, language impairment, motor function deficits, and seizures. More than 90% of patients with this disorder have a missense mutation within or adjacent to the nuclear localization signal (NLS) of hnRNPH2, although the specific pathogenic consequences of these mutations have not been examined. Here we found that hnRNPH2 NLS mutations result in reduced interaction with the nuclear transport receptor Kap{beta}2 in vitro and in cultured human cells. These mutations also cause modest accumulation of hnRNPH2 in the cytoplasm, suggesting that mislocalization of the protein might contribute to pathogenesis. We generated two knock-in mouse models with human-equivalent mutations in the endogenous mouse gene Hnrnph2, as well as Hnrnph2 knockout (KO) mice, and subjected them to extensive phenotyping. Mutant knock-in mice displayed a spectrum of phenotypes that recapitulated aspects of the human disorder, including reduced survival in males, craniofacial abnormalities, impaired motor and cognitive functions, and increased susceptibility to audiogenic seizures. Mutant knock-in male mice developed more severe phenotypes than female mice, likely due to differences in X-chromosome gene dosage. In contrast, two independent lines of Hnrnph2 KO mice showed no detectable phenotypes. Notably, KO mice had upregulated expression of Hnrnph1, a close paralog of Hnrnph2, whereas mutant Hnrnph2 knock-in mice failed to upregulate Hnrnph1. Thus, genetic compensation by Hnrnph1 might be sufficient to counteract the loss of hnRNPH2. These findings suggest that the pathogenesis of HNRNPH2-related disorder in humans may be driven by a toxic gain of function or a complex loss of HNRNPH2 function with impaired compensation by HNRNPH1. The mutant knock-in mice described here are an important resource for preclinical studies to assess the potential benefit of either gene replacement or therapeutic knockdown of mutant hnRNPH2.

neuroscience↗