Nature Structural & Molecular Biology

Linker histones are essential for chromatin compaction, yet how they contribute to higher-order fiber assembly remains poorly understood. Here, we determined cryo-electron microscopy structures of Arabidopsis dodeca-nucleosome fibers containing distinct H2A/H3 variants and linker histone H1.3, revealing a noncanonical binding mode that a laterally positioned H1.3 connects the acidic patch of one nucleosome and the DNA of the neighboring nucleosome, thereby scaffolding dinucleosomes into two-start chromatin fibers. This noncanonical binding mode is structurally conserved when H1.3 is replaced by Gallus gallus H5. Furthermore, incorporation of H2A.W and H3.3 further induces back-to-back fiber dimerization. Cryo-electron tomography and in vivo cross-linking mass spectrometry analyses support the physiological relevance of H1 lateral engagement. Our findings establish that linker histones act as active architectural scaffolds in higher-order chromatin organization.

Pre-mRNA splicing determines the expressed proteome and is frequently dysregulated in cancer. The tumour-suppressor RBM5 controls an exon network regulating apoptosis, yet its molecular mechanism is elusive. Using in vivo spliceosome capture and cryogenic electron microscopy, we determined structures of precatalytic spliceosomes arrested by RBM5 immediately after U2 snRNP branchpoint recognition. Despite intron diversity, the U2-pre-mRNA duplex, branchpoint adenine, and downstream polypyrimidine tract are well-resolved. RBM5 binds the outer SF3B1 HEAT surface and performs dual functions: First, its helix-loop-helix motif and upstream zinc-finger domain sterically block tri-snRNP and Prp8 docking and prevent progression to pre-B and Bact complexes; Second, its G-patch activates DHX15 and places this DExH-box helicase on the pre-mRNA as it exits SF3B1, poised for branch helix unwinding. DHX15 binding to SF3B1 is facilitated by U2SURP/SR140, which engages SF3B1 near RBM5s helix-loop-helix. Functional assays confirm that disruption of the RBM5 interfaces with either DHX15 or SF3B1 inhibit exon repression. Mutations at these regulatory interfaces are common in cancer genomes and predicted to disrupt its regulation of apoptotic isoforms. Thus, RBM5 acts as a dual-action spliceosome gatekeeper that couples helicase activation with physical stalling to enforce tumour-suppressive alternative splicing programmes.

The cyclin-dependent kinase CDK11 functions in transcription, mitotic progression, and mRNA splicing. Specifically, spliceosome activation during the B to Bact transition depends on phosphorylation of the U2 snRNP component SF3B1 by the CDK11-cyclin L-SAP30BP complex. Here, we present the structure of this spliceosome-activating CDK-cyclin complex, determined by cryogenic electron microscopy at 2.3 [A] resolution. Our structure and biochemical experiments show that SAP30BP forms extensive interactions with cyclin L2, thereby stabilising it, and forms critical interactions with the C-terminal kinase lobe of CDK11 that promote complex assembly. Destabilisation of cyclin L2 in the absence of SAP30BP suggests that these principles are applicable to all CDK11-cyclin L complexes. Furthermore, we identify a pseudo-substrate sequence near the CDK11 C-terminus and provide evidence for a role of this segment in CDK11 auto-regulation. Finally, the structure of the CDK11-cyclin L2-SAP30BP complex bound to the clinical high-affinity CDK11 inhibitor OTS964 and a comparison to OTS964-bound off-target complexes provide insight into the mechanism of OTS964 selectivity and specificity.

Cargo-specific activating adaptors enable dynein to assemble with dynactin into processive supercomplexes. Adaptors share a coiled-coil architecture, but are highly diverse in sequence and structure, raising the question of how they converge on a common activation mechanism. To address this, we determined near atomic cryo-EM structures of dynein-dynactin assembled with five adaptors: RAB11FIP3, NIN, TRAK1, BICD2 and HOOK3. Despite their heterogeneity, all complexes contain adaptor coiled coils which bridge two dynein dimers to the dynactin filament. Adaptors are defined by an N-terminal interaction at the HBS1 with the dynein heavy chain, additional contacts along the dynein-dynactin groove, and C-terminal binding to the dynactin pointed end. However, we also found distinct sequence features, coiled-coil breaks and pointed-end interfaces that tune complex stoichiometry and stability. Our results define shared principles of dynein activation while revealing unexpected plasticity in how adaptors recognise and organise the dynein-dynactin machinery.

In Caenorhabditis elegans, the condensin IDC complex represses transcription from both X chromosomes in hermaphrodites to achieve dosage compensation. How condensin IDC is specifically recruited to the X chromosomes in coordination with sex determination and dosage compensation (SDC) proteins and how it modulates gene expression have, however, remained unresolved. Here, we identify SDC-3 as the key adaptor that directly binds the elbow coiled-coil domain of the condensin IDC-specific SMC subunit DPY-27. Using cryo-electron microscopy, we determine the structure of the SDC-3 adaptor domain bound to an auto-inhibited condensin IDC holoenzyme. Disrupting this interaction compromises dosage compensation and diminishes condensin IDC enrichment on the X chromosomes. Upon overcoming auto-inhibition, condensin IDC exhibits robust DNA loop-extrusion activity comparable to that of canonical condensin. We propose that SDC-3-anchored condensin IDC exploits loop-extrusion to reorganize X-chromosome chromatin and mediate chromosome-wide transcriptional repression.

Eukaryotic mRNAs are co-transcriptionally capped at the 5' end with a methylated m7G moiety (cap0)1, which in higher eukaryotes is further methylated on the ribose (Nm) of the transcription start site (TSS) nucleotide to create the cap1 structure (m7GpppNm). Coronaviruses that replicate in the cytoplasm encode their own capping enzymes to acquire this cap1 structure which facilitates translation and shields them from the host innate immune system2-5. Here we report the identification of an additional N6-methyladenosine (m6A) methylation on the 5' cap (m7Gpppm6Am) of the human coronavirus SARS-CoV-2 RNA. It is catalysed by the host m6A methylase PCIF16-9 following capping by virus-encoded non-structural protein NSP 14 and NSP1610. Human cell cultures lacking PCIF1 accumulate reduced levels of the viral RNA and support reduced viral replication. Furthermore, Pcif1 mutant mice infected with SARS CoV-2 display milder symptoms. We identify the host RNA methyltransferase PCIF1 as a critical ally of SARS CoV-2 for viral replication.

Upon DNA damage, chromatin remodeling is rapidly initiated to promote chromatin accessibility, thereby facilitating the recruitment and assembly of repair factors. Although this enhanced accessibility has been linked to poly(ADP-ribose) polymerase (PARP) activity, the mechanism by which cells overcome the nucleosome barrier remains unclear. Using our designer chromatin system, we uncovered a previously uncharacterized activity of PARP1, whereby it directly and asymmetrically evicts histone dimers proximal to DNA strand breaks from nucleosomes to generate oriented hexasomes. In the presence of HPF1, PARP1 generates stable PARylated hexasomes, an open chromatin intermediate that can serve as a bifunctional hub for recruitment of DNA- and PAR-dependent factors. Using cellular assays, we demonstrated that PARP activity is both required and sufficient to drive chromatin accessibility and the recruitment of repair factors, with direct involvement of subnucleosomal species. Unexpectedly, we identified the C-terminal tail of histone H2A, a motif harboring recurrent cancer-associated mutations, as a critical determinant of efficient PARP1-mediated nucleosome disassembly. Deletion of the H2A tail sensitizes cells to DNA-damaging agents and PARP inhibitors, implicating a functional role of PARP1-mediated nucleosome disassembly in DNA repair. Together, our findings support a model in which PARP1 directly drives histone eviction, leading to the formation of subnucleosomes that facilitate efficient DNA repair.

RNA 5-methylcytidine (m5C) is a prevalent modification that drives RNA stability and function. In humans, m5C is deposited on distinct RNA substrates by DNMT2/TRDMT1 and the NSUN family, to regulate diverse cellular processes, but how m5C writers recognise their substrates remains unclear. NSUN2 is a major m5C methyltransferase with broad roles in cell physiology and strong links to cancer and neurodevelopmental disorders 1. Here, we reconstitute an active human NSUN2-tRNA complex and capture its post-catalytic, tRNA-bound structure at 3.1 [A] resolution. Using an integrated approach combining biochemistry, cryo-electron microscopy, crosslinking mass spectrometry and molecular dynamics simulations, we show that NSUN2 remodels the tRNA to access the variable-loop target cytidine. Recognition is driven by RNA architecture, with NSUN2 exploiting the L-shaped tRNA scaffold to position the target base in the catalytic centre. We further show that Gly679 at the NSUN2-tRNA interface is important for the stability of the complex, providing a mechanistic basis for how the disease-associated Gly679Arg substitution can impair tRNA binding. Together, these findings establish an RNA-structure-guided mechanism for NSUN2 substrate recognition and methylation and provide general principles for m5C deposition on cellular RNAs and their fundamental role in disease.

In meiosis, crossovers between homologous chromosomes generate genetic diversity and are required for accurate chromosome segregation, ensuring fertility. In mammals, HEI10 is one of three pro-crossover RING-domain factors implicated in protein modification by ubiquitin and/or SUMO and characterised by their dynamic accumulation at future crossover sites. However, the molecular architecture and enzymatic activity of mammalian HEI10 have remained unknown. Here, we show that human HEI10 has E3-ubiquitin ligase activity that depends on its higher-order assembly. We report the crystal structure of the HEI10 core, revealing how a 29-nm rod-like tetramer is formed through head-to-head association of two coiled-coil dimers that results in clustering of four RING domains around the molecular centre. HEI10 tetramers self-assemble through RING, coiled-coil, and C-terminal interfaces into fibrous and spherical higher-order structures. Structure-guided mutants show that higher-order assembly is required for HEI10 to catalyse K63-linked ubiquitin chain formation in vitro, with the most active species likely corresponding to a loose, non-fibrous network of assembled HEI10 molecules. Arabidopsis thaliana HEI10 retains the tetrameric core and higher-order assembly behaviour, suggesting a conserved principle of HEI10 function.

Proteasome activators (PAs) bind the -subunits of the human 20S proteasome (h20S), opening the "gates" and allowing entry of substrate proteins. Aging is associated with diminished proteasome activity, leading to interest in understanding this activation mechanism. Evolving models have been proposed regarding PAs C-terminal tails, yet the critical molecular contacts for gate-opening are unclear. Here, we show a conserved leucine in the 5th position (P5) of the C-terminus is essential for h20S gate opening. By engineering C-termini in a model activator, PA26E102A, we show mutations to P5 systematically modulate proteasome activity in vitro and in cells. Structures of PA26E102A:h20S complexes at 2.7-3.2 [A] resolution identify interactions between P5 and a conserved arginine in the h20S, leading to partial or full gate opening. These results clarify the essential contacts required for h20S gate opening, potentially enabling design of proteasome activation therapies.

We report the discovery and structure of a previously unknown ~1 MDa hollow protein assembly, identified during a survey of ciliary complexes from the ciliate Tetrahymena thermophila. By combining mass spectrometry, structure prediction, and cryo-electron microscopy, we define a homotetrameric cage-like complex with a distinctive elliptical architecture and a large internal cavity. A sequence survey revealed several thousand homologs spanning diverse unicellular eukaryotes--including green algae, fungi, amoebozoans, choanoflagellates, and SAR lineages--as well as predominantly gram-negative bacteria, indicating an ancient evolutionary origin and arguing against a eukaryote-specific function. We determined a near-atomic resolution structure of the complex from the slime mold Dictyostelium discoideum, demonstrating conservation of overall architecture and cavity despite low sequence identity. Together, these results establish the CAGE complex (Conserved Assembly in Gram-negative bacteria and Eukaryotes) as a new class of large protein cage broadly distributed across the tree of life. While its biological function remains unknown, its size, architecture, and conservation suggest possible roles in transport or protein/RNA homeostasis.

At the endoplasmic reticulum (ER), membrane protein quality control is tightly regulated to ensure excess subunits are recognized and degraded to protect cellular homeostasis. Using genome wide CRISPR screens, we identified a factor of unknown function, thioredoxin domain containing protein 15 (TXNDC15), and showed that it regulates membrane protein stability by tuning the activity of the E3-ubiquitin ligase, MARCHF6. TXNDC15 modulates MARCHF6 in two opposing ways: first, it enhances the binding, ubiquitination, and degradation of membrane protein subunits with soluble cytosolic domains; and second, it prevents the inappropriate recruitment and ubiquitination of subunits with globular lumenal domains. Patient mutations to TXNDC15 that cause the ciliopathy Meckel-Gruber syndrome, disrupted its binding to MARCHF6, allowing degradation of critical ciliary proteins as they transit through the ER leading to defects in ciliogenesis. The regulatory function of TXNDC15 therefore exemplifies how protein quality control maintains the integrity of the proteome to prevent disease.

P2X receptors are trimeric ATP-gated ion channels that assemble as homo- or heterotrimers, with heteromeric forms exhibiting intrinsic asymmetry that influences function. Here, we report four high-resolution cryo-EM structures of human P2X2/3 heterotrimers representing distinct functional states, including ATP-bound assemblies (P2X332 and P2X223), the apo form, and a ligand/ATP-bound closed conformation. The three ATP-binding sites show asymmetric recognition of MgATP{superscript 2}- and ATP-, and channel activation requires occupancy of only two MgATP{superscript 2}- molecules. Gefapixant binds a single allosteric site and selectively inhibits MgATP{superscript 2}-, but not ATP-, binding, indicating orthosteric-allosteric coupling within the heterotrimer. Structural features of the transmembrane domain define ion permeation, particularly for Ca{superscript 2}. Despite asymmetric ligand interactions, gating remains largely symmetric, with minor differences in desensitization. These findings provide a structural framework linking asymmetry to coordinated channel function and open avenues for subtype-selective therapeutic intervention.

The restoration of chromatin in the wake of a DNA or RNA polymerase is essential to maintain the integrity of eukaryotic genomes. Human HIRA is a 1.8-megadalton, three-subunit histone chaperone that mediates all replication-independent deposition of the histone variant H3.3 at active chromatin regions1-6. Disruption of HIRA perturbs active-chromatin organization and has wide-ranging consequences for development, cellular senescence, and genome integrity7-11. Despite its central biological role in reassembling nucleosomes post-transcription, the structure of native human HIRA and the mechanism by which it organizes histones and DNA during nucleosome assembly remain unknown. In particular, the function of the largest HIRA subunit CABIN1 is enigmatic. Here, we show that HIRA is not simply a passive histone hand-off factor but remains engaged across multiple stages of nucleosome assembly, including a close interaction with the nucleosome. Cryo-EM structures reveal that HIRA forms an extended arch-like structure that binds the nucleosome primarily through extensive CABIN1 contacts with histones, histone tails, nucleosomal DNA, and linker DNA, during the final stage of nucleosome assembly. Together, our results suggest a testable mechanism for HIRA-mediated nucleosome assembly and product release and provide the basis for elucidating the molecular details of this fundamental biological process.

Proper polyadenylation site (PAS) selection is critical for RNA isoform determination. Core spliceosomal components, including U1 snRNP, regulate PAS choice, but whether they work with other splicing factors in this role remains unclear. Here, we establish that the splicing factor SRSF1 regulates PAS selection independently of and through interactions with U1 snRNP. Independent of U1 snRNP, SRSF1 binds RNA near proximal PASs within 3 UTRs to promote their usage, and, in line with this observation, breast cancer tumors with altered SRSF1 levels display shifted 3'-end selection. In conjunction with U1 snRNP, SRSF1 acts on PASs through U1 snRNP-mediated SRSF1-Pol II interactions. Consistent with co-transcriptional regulation, SRSF1 reduces the Pol II elongation index and limits transcription readthrough. Together, our results reveal that SRSF1 shapes RNA isoform determination beyond its canonical role in splicing, through a combination of direct RNA binding and U1 snRNP-dependent coordination with Pol II.

The receptor tyrosine kinase RET is activated by GDNF and GFR1 together as a bipartite ligand, driving receptor activation and signalling in developmental and neuroprotective contexts. Evidence from developmental and cell models has suggested that heparan sulfate (HS) functions as a fourth component in RET signalling by binding to GDNF, but the molecular details remain unclear. Here, we present the cryo-EM structure of the heterohexameric RET:GDNF:GFR1 complex with a fully resolved heparin ligand, revealing an unexpected extended HS binding site spanning all three proteins. The architecture of the complex and binding mode of the HS chain in this complex enables the formation of a higher order 4:4:4 assembly bound to a single 30-saccharide HS chain which bridges two intimately bound complexes. This multi-protein interface selectively binds the highly sulfated domains of HS over other GAG classes, and is essential for RET activation in trans with soluble GFR1, but not in cis when GFR1 is membrane-bound. Our data suggest that HS shapes the dynamics of RET signalling at every stage, from ligand diffusion to signalling complex formation. Thus, GDNF-GFRa1 paracrine signalling reveals a surprising dependence for long-chain GAG function in which the glycan engages in both receptor complex assembly and clustering.

YicC is a conserved protein, recently discovered to have hydrolytic endoribonuclease activity, with a phylogenetic distribution implicating an ancestry deeper than that for most extant ribonucleases central to RNA metabolism. We present evidence that Escherichia coli YicC can cleave the small regulatory RNAs (sRNAs), RyhB and RprA, even when they are sequestered in otherwise protective complexes with the RNA chaperone Hfq. Nonetheless, YicC activity is markedly diminished when RyhB and other sRNAs are paired with cognate mRNA. Our cryoEM structures of catalytically inactive YicC in complex with RyhB and in the apo state reveal quaternary and tertiary structural switches, triggered by substrate engagement, that engulf and ratchet a stem loop element of the sRNA into an internal channel, where metal-assisted hydrolytic action occurs. Based on these findings, we propose that the enzyme favours specific stem-loop structures and may discriminate between pools of active, target-engaged sRNAs and those that are inactive. The mechanism for substrate-triggered conformational switching could represent an ancient strategy for selective RNA degradation.

Allosteric modulators offer opportunities for pathway-selective GPCR signalling, but the structural mechanisms enabling biased allosteric modulation remain unclear. Here we identify a helix 8-proximate allosteric site in the immune-metabolic receptor GPR84 and define how it achieves Gi -biased signalling. Cryo-EM structures of the GPR84-Gi complexes bound to the orthosteric agonist OX04539 alone or in combination with the positive allosteric modulator (PAM) PSB-16671 reveal that PSB-16671 binds at the interface of TM1, TM7, and helix 8, a location distinct from previously characterized GPCR allosteric pockets. Molecular dynamics simulations and mutagenesis uncover a polar interaction network linking orthosteric and allosteric sites through conserved residues including Asp662.50, Asn1043.36, and Asn3627.45. Unexpectedly, disrupting this network enhances allosteric cooperativity, indicating that conformational flexibility within the network is essential for allosteric communication. PSB-16671 stabilizes a receptor conformation with pronounced TM6 displacement that favours Gi coupling while disfavouring {beta}-arrestin recruitment. This Gi-biased profile sustains macrophage phagocytosis of cancer cells without the desensitization induced by balanced agonists. Sequence analysis suggests that helix 8-proximate allosteric sites may be broadly targetable across class A GPCRs, while receptor-specific contacts enable selective modulation. These findings establish structural and mechanistic principles for biased allosteric modulation applicable beyond GPR84.

The RAS-RAF-MEK-ERK cascade regulates critical cellular processes such as cell proliferation and differentiation, with its dysregulation driving over 30% of human cancers. However, the active state of MEK and molecular mechanisms of ERK phosphorylation remain unclear. Here, we report the cryo-EM structures of phosphorylated MEK1 (pMEK1) bound to unphosphorylated and monophosphorylated ERK1. These structures reveal that pMEK1 interacts with ERK1 via three interfaces: first between MEK1 N-terminal peptide and ERK1 D-site recruitment site (DRS), second between MEK1 C-lobe and ERK1 F-site recruitment site (FRS), and third between MEK1 activation loop and ERK1 activation loop. Notably, we identify an unexpected phosphorylation mechanism: pMEK1 catalyzes ERK1 T202 phosphorylation through phosphate transfer from ERK1 Y204. In addition, pMEK1 exhibits phosphatase activity, catalyzing the dephosphorylation of ERK1 Y204. These findings allow us to delineate the catalytic cycle of MEK-catalyzed ERK phosphorylation and provide insights for targeting this oncogenic pathway.

RAF kinases activate MEK in the RAS-MAPK signaling pathway, and changes in RAF kinase signaling have been linked to tumor formation. RAF1 requires the HSP90-CDC37 chaperone system for proper activation, but how the HSP90-CDC37 chaperone system regulates RAF kinase maturation remains enigmatic. We present novel cryo-EM structures of previously uncharacterized RAF1 chaperone complexes, including a 2:2:2 RAF1-HSP90-CDC37 complex (RRHCC), intermediate assemblies (RHCC), and a RAF1-HSP90-CDC37-p23 complex (RHCp23). These reveal an asymmetric stepwise folding mechanism unique among HSP90 kinase clients in which one RAF1 threads through the HSP90 lumen while another is captured in a "casting mold" formed by CDC37 and HSP90 that stabilizes the partially folded C helix of RAF1. The RHCp23 structure shows how p23 cooperates with CDC37 to regulate ATP hydrolysis and client release. The HSP90-CDC37 system supports pre-dimerization of RAF1 and BRAFV600E homodimers and RAF1 heterodimers, a mechanism unique to RAF among kinase clients of HSP90. Phosphoproteomics reveals selective activating phosphorylations within RRHCC. These RAF isoform complexes differentially activate MEK signaling and cell proliferation, establishing HSP90-CDC37 as not just a passive stabilizer but an active regulator of RAF signaling with therapeutic implications.