Nature Structural & Molecular Biology

Dihydroorotate dehydrogenase (DHODH), which catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, is a validated therapeutic target in cancer, autoimmune disorders and infectious diseases. DHODH is hypothesized to utilize a ping-pong catalytic mechanism. However, available crystal structures of DHODH in complex with small molecule inhibitors are irreconcilable with this model, showing simultaneous occupancy of both substrate binding sites. To elucidate the structural basis for DHODH-mediated catalysis, we resolved the structures of two DHODH holoenzymes. These structures capture novel conformational states that show mutually exclusive substrate binding. The holoenzyme structures also suggest that conformational changes in the catalytic loop of DHODH play a critical role in regulating enzyme activity. To map the functional landscape underlying DHODH inhibitor resistance, we performed deep mutational scanning drug-resistance screens with two clinically-relevant structurally distinct DHODH inhibitors, BAY2402234 and brequinar. Resistance variants cluster in the inhibitor binding site and at a previously unappreciated surface pocket on DHODH that allosterically regulates ubiquinone binding. Together, our findings provide structural evidence for the ping pong model of catalysis, define the mutational landscape governing inhibitor resistance, and reveal novel structural vulnerabilities in DHODH that can be utilized for future drug development.

In the paper by Nguyen et al, published in Nature Communications in 2024, we describe structural polymorphism in fibrils extracted from patients with ATTR amyloidosis. ATTR amyloidosis is characterized by deposition of transthyretin amyloid fibrils across multiple organs, resulting in marked phenotypic variability. Previous studies on ATTR suggest its phenotypic variability may be linked to differences in fibril structure, as demonstrated in amyloid diseases such as AA amyloidosis, tauopathies, or synucleinopathies. Thus far, most ATTR fibrils adopt a strikingly similar fold across patients and affected organs. The most notable exception is described in the study by Nguyen et al., where we report the identification of two co-existing polymorphs in cardiac fibrils from three patients with the ATTRv-I84S variant. These cardiac polymorphs differ in conformation of the segment Gly57 to Gly67, termed the gate, which regulates access to a central polar channel within the fibril core. In one polymorph, the gate adopts an open conformation, exposing the channel, whereas in the other it is closed, shielding it. Whether these polymorphs extend beyond cardiac tissue remains unknown. Here, we address this question by extending our analysis to additional organs from a fourth patient with ATTRv-I84S amyloidosis.

The short arms of human acrocentric chromosomes are characterized by nucleolar organizer regions essential for ribosome biogenesis, but their highly repetitive nature has hindered genomic analysis. Leveraging the recently completed genomes of all major great ape lineages, we identified recurrent features of their acrocentrics, including enriched repeat classes, centromere repositioning by whole-arm inversion, interchromosomal sequence exchange, and birth-and-death evolution of multiple gene families under selection. Together, these processes have enabled the repeated amplification and diversification of the FRG1 gene family over 25 million years of ape evolution, and, in gorilla, the amplification of a novel IGSF3-GGT fusion gene. Similar evolutionary events also explain the distribution of segmental duplications and heterochromatin in the modern human genome, predisposing it to karyotypic abnormalities such as Robertsonian translocations. Our findings highlight acrocentric chromosomes as key drivers of evolution in the great apes, with implications for speciation, adaptation, and clinical genomics.

Eukaryotic mRNAs carry a 5'-terminal 7-methylguanosine (m7G) cap that is recognized by multiple cellular proteins and is essential for mRNA self-discrimination, stability, and translation. The eukaryotic translation initiation factor 4E (eIF4E) binds the 5' cap and promotes recruitment of mRNAs to the ribosomal translation initiation complex, a critical step in cap-dependent translation. Beyond naturally occurring cap modifications that modulate mRNA interactions and fate, non-natural 5' cap modifications can be used to finely tune diverse aspects of mRNA biology. This has gained particular relevance with the emergence of in vitro transcribed mRNAs as a powerful therapeutic modality for protein replacement therapies and vaccines. Here, we report binding data for a series of synthetic 5' cap analogues interacting with eIF4E, together with X-ray crystal structures of eIF4E-cap complexes, providing detailed insights into the structural determinants of cap recognition by eIF4E. Furthermore, analysis of gene expression from mRNAs carrying different synthetic cap analogues in human cell lines and mice revealed a relationship between cap architecture and protein expression. Taken together, our structural, biochemical, and functional data establish structure-activity relationships for synthetic cap analogues and identify structural features associated with enhanced translational performance.

Variation alters inherent protein properties like thermodynamic stability and catalytic activity, and these intrinsic effects are shaped by the complex cellular environment. Signaling cascades typify this complexity, with tens or hundreds of proteins acting in concert through regulated states and interactions to transduce signals. Here, we measure the effect of 186,676 variants across 17 proteins in the RAS-MAPK pathway on protein abundance and pathway activity in human cells under diverse conditions, for a total of 503,099 variant effect measurements. We discovered 14,412 dominant negative variants across 12 proteins, nearly all previously unknown, that disrupt specific domains or interaction interfaces while leaving other functions intact and that are depleted from the human population. Measuring the abundance of 106,642 variants across 10 proteins under HSP90 inhibition showed that HSP90 shapes the effect of 67% of missense variants across the pathway, buffering destabilizing variants in both strong and weak clients. Our variant effect data, collected in the context of an intact proteome, enabled us to assess the functional importance of 395 experimentally and computationally modeled MAPK protein interactions. Using additional mutational scans, we resolved how the promiscuous switch II helix binding surface of KRAS accommodates overlapping interactions with LZTR1 and SOS1, revealing distinct allosteric networks that support each interaction. Thus, we establish a framework for measuring variant effects at unprecedented scale in human cells, illuminating fundamental MAPK pathway signaling mechanisms and demonstrating that cellular context profoundly shapes the effect of most variants.

Nucleosome positioning plays a fundamental role in regulating DNA accessibility and cellular activity, yet the precise rules by which mammalian sequences instruct this organization have remained incomplete. Prior work has established that intrinsic sequence preferences dictate nucleosome architecture in yeast, but in mammals, this sequence code is modulated by chromatin state and cell-type-specific factors. Here, we train a deep-learning model on genome-wide maps from mouse embryonic stem cells to predict nucleosome occupancy directly from the primary DNA sequence. Our model accurately predicts in vivo occupancy and captures shared biological signals while effectively denoising assay-specific technical biases. Through in silico mutagenesis, we identify specific nucleosome positioning regions comprised of short 5-to-20-base-pair motifs concentrated within dense regulatory windows. Furthermore, we demonstrate that repetitive DNA elements play a major role in establishing these highly organized nucleosome arrays on a genomic scale. Specifically, CCCTC-binding factor motifs embedded within short interspersed nuclear elements, zinc-finger-bound motifs within long interspersed nuclear elements, and microsatellite repeats act as potent organizers of nucleosome phasing. Together, our findings extend the concept of sequence-encoded nucleosome architecture to mammalian genomes and reveal that regularly spaced nucleosome islands are shaped by short, recurrent sets of DNA motifs that couple local sequence to chromatin structure.

Advances in protein structure prediction have enabled all-atom protein-ligand co-folding models that predict bound conformations directly from sequence and small-molecule structure. However, these models often fail to generalize to novel binding sites or alternative protein conformational states, limiting their utility for chemical biology and drug discovery. Here we show this limitation reflects training data bias rather than architectural constraints and can be overcome through targeted finetuning. Using ten previously unseen X-ray structures of Werner (WRN) helicase from a drug discovery program, we finetune Boltz-1 to learn both an allosteric binding site and a large conformational change locking the enzyme in an inactive state, while preserving accuracy on the ATP-bound state. The finetuned model generalizes to different chemical series and transfers the conformational logic across RecQ-family helicases in a binding-site sequence-dependent manner. This approach provides a blueprint for adapting foundation models as new structural and mechanistic data emerge, enabling co-folding networks to capture ligand-induced conformational switches and binding poses absent from their training data but central to biological regulation and therapeutic intervention.

Nucleosomes regulate chromatin folding, accessibility, and factor recruitment. Current models primarily attribute these functions to histone tail modifications, while the core is largely viewed as a structural scaffold. Yet subtle changes within the nucleosome core can produce profound functional consequences, and the mechanisms underlying these effects remain unclear. Here, we describe nucleosome core allostery as a fundamental principle of chromatin regulation that amplifies the impact of minimal nucleosome variations. Leveraging natural differences between H2A.Z variants, we show that the nucleosome core encodes distinct conformational dynamics that propagate allosterically, thereby controlling nucleosome accessibility and recognition by chromatin factors. As a result, a single buried amino acid substitution alone is sufficient to reprogram nucleosome dynamics and bias cell identity. Our findings establish the nucleosome core as an allosteric regulatory module and provide a generalizable framework for how subtle variation within nucleosomes is amplified into diverse biological outcomes in development and disease.

The targeting function of nuclear localization signals (NLSs) is generally considered independent of a protein's native sequence or fold and is readily transferable to heterologous cargos. Contrary to this paradigm, rapid nuclear translocation of phosphorylated STAT1 (pSTAT1) following cytokine stimulation requires importin {beta}, Ran-GTP, and the importin 5 isoform, which recognizes a non-transferable NLS. Here, we present cryo-EM structures of pSTAT1 bound to importin 5, revealing an asymmetric 2:1 complex that diverges from canonical NLS-mediated cargo recognition. Importin 5 occupies the DNA-binding groove of the pSTAT1 dimer, with a single STAT1 N-terminal domain positioning the C-terminal Armadillo repeats 9-10 (S1B domain) orthogonal to the DNA-binding interface. This interface is also targeted by the Ebola virus protein VP24, an antagonist of interferon signaling. We further show that Ran-GTP alone is insufficient to trigger nuclear release of pSTAT1, which additionally requires the exportin CAS. A cryo-EM reconstruction of the CAS-Ran-GTP-5 complex, supported by in vitro competition assays, demonstrates that CAS and pSTAT1 are mutually exclusive ligands for importin 5. Together, these findings define the molecular choreography of cytokine-induced STAT1 nuclear translocation and release, establishing a general paradigm for STAT family signaling.

Regions of transcription initiation are often enriched in the histone variant H2A.Z. This is especially true in the divergent eukaryote Trypanosoma brucei, where broad domains of H2A.Z and a kinetoplastid-specific variant H2B.V define a limited number of polycistronic transcription start sites. To explore the chromatin underpinning this unusual transcription, we reconstituted H2A.Z-H2B.V nucleosomes in vitro and found that they are inherently unstable, form open chromatin structures, and serve as a direct interaction scaffold for gene regulators, in line with their role in transcription activation. A single particle cryo-EM structure reveals why H2A.Z-H2B.V form obligate dimers, pack differently within nucleosomes, and exhibit altered binding to DNA. We identified unique features of H2A.Z and H2B.V that result in differential activity of chromatin modifying enzymes, including the H2A.Z C-terminal tail and an atypical acidic patch. An affinity purification mass spectrometry screening approach revealed that H2A.Z-H2B.V nucleosomes have a distinct protein interaction profile, suggesting that interactions with chromatin factors are directly tuned for transcription control. Overall, this work reveals how trypanosome-specific histone features both inherently alter the biomechanical properties of nucleosomes as well as reshape chromatin binding and modification patterns at transcription start sites.

Topoisomerase cleavage complexes (TOPcc) are covalent protein-DNA intermediates formed transiently during topoisomerase catalysis and constitute a major class of DNA-protein crosslinks in human cells, yet their endogenous genomic distributions remain difficult to resolve. Here, we developed protein-DNA adduct immunoprecipitation followed by sequencing (pDAIP-seq), a highly sensitive method for detecting endogenous TOPcc that enables their genome-wide profiling under normal physiological conditions without stabilization by topoisomerase poisons. Using pDAIP-seq, we found that camptothecin treatment, required by existing TOP1cc profiling methods, not only increased TOP1cc abundance but also substantially altered its genomic distribution. Endogenous TOP1cc were globally reduced upon transcriptional suppression and increased upon TOP2A depletion, supporting TOP1cc regulation by the maintenance of genome-wide DNA topological homeostasis. Interestingly, both the regional distribution and nucleotide-scale positioning of TOP1cc, including the positions of TOP1 catalytic cleavage sites, were largely specified by local DNA sequence context. By extending pDAIP-seq to capture transient covalent protein-DNA intermediates involved in DNA repair, including TDP1-, PARP1-, and KU70-DNA adducts, we further identified endogenous TOP1cc as a potential major source of spontaneous DNA breaks in human cells. Finally, pDAIP-seq also enabled genome-wide mapping of TOP2cc, TOP3cc, and SPO11-DNA covalent intermediates in mouse testis undergoing programmed meiotic DNA cleavage. Together, these findings establish pDAIP-seq as a broadly applicable method for the mapping of covalent protein-DNA intermediates and reveal endogenous TOP1cc as a key intermediate between DNA topological regulation and human genome instability.

The LINC (linker of nucleoskeleton and cytoskeleton) complex is a central mechanotransductive apparatus that physically couples cytoskeletal forces to the nuclear interior, thereby regulating nuclear architecture, chromatin organization, and downstream signaling. Using atomistic molecular dynamics simulations, we identify and characterize force gates within the LINC complex, structural interactions that maintain mechanical connectivity under load. By introducing targeted mutations into the KASH peptide and SUN protein, we provide computational evidence that a key disulfide bond (SUN2 Cys563 to KASH2 Cys6862) and specific residues within the KASH {beta} strand interaction motif (KASH6875, KASH6877) act as distinct mechanical elements during force transmission across the SUN and KASH interface. Leveraging this framework, we show that the disulfide bond and the {beta} sheet network contribute unequally: removing the disulfide reduces both sustained and peak force transmission approximately twofold, whereas disrupting the {beta} sheet network leaves peak force largely intact while altering interfacial coupling and the duration over which load is borne. Localized molecular changes therefore reshape mechanical coupling to the nucleus through distinct routes rather than through a single graded loss of force. Such altered LINC mechanics may be relevant to laminopathies, including Hutchinson Gilford Progeria Syndrome (HGPS), in which defective nuclear lamina compromises force transmission; however, implications for specific diseases remain speculative and require experimental validation. By resolving how distinct molecular contacts contribute to force transmission in space and sequence, this work establishes a framework for dissecting mechanical coupling at protein interfaces under load. These findings provide a foundation for future studies aimed at experimentally testing how individual interface interactions govern nuclear force transmission, a prerequisite for therapeutically targeting LINC mechanics. More broadly, the force gate framework may guide strategies to modulate interfaces that bear mechanical loads in other mechanically regulated proteins. Together, these findings establish a computational framework for investigating force transmission at mechanically loaded protein interfaces and provide experimentally testable predictions for future studies.

Transient receptor potential melastatin 3 (TRPM3) is a calcium-permeable ion channel implicated in chronic pain and developmental and epileptic encephalopathies. The voltage sensor-like domain (VSLD) is a major site of pharmacological modulation, but the molecular determinants of ligand recognition and efficacy remain incompletely understood. Here we combine cryo-electron microscopy, molecular dynamics simulations, mutagenesis, and medicinal chemistry to investigate the interactions of TRPM3 with the inhibitors isosakuranetin, maprotiline, and diclofenac. We show that structurally diverse ligands occupy a common VSLD cavity but engage distinct interaction networks. Functional and computational analyses identify ligand-specific residue dependencies and support a halogen bond between diclofenac and D947 that contributes to inhibitor potency. Furthermore, esterification or amidation of the diclofenac carboxylate converts inhibition into potentiation, and mutagenesis and molecular dynamics simulations support partially overlapping VSLD configurations associated with these opposing effects. Together, these findings establish the TRPM3 VSLD as an adaptable allosteric pocket in which ligand-specific interactions shape recognition, potency, and efficacy.

Multidrug-resistant pathogens demand novel resistance-breaking strategies. The oligoarylamide albicidin and pyrrolobenzodiazepines (PBDs) are potent antibacterials with unrelated scaffolds, yet both are neutralized by albA gene products in many Gram-negatives: AlbA, an albicidin-responsive self-upregulating MerR-family factor, and the smaller AlbAS, its unique ligand binding domain (LBD) alone. How AlbA couples ligand sensing to transcriptional activation has remained elusive. Here, using an integrative multidisciplinary approach, we define this mechanism and show that albicidin and PBDs elicit different responses. Crystal structures reveal C8-linked PBDs bound to AlbAS at the N- and C-terminal subdomains (NTD, CTD) of the central tunnel in poses dictated by C8-tail chemistry. This plasticity sequesters diverse PBDs with nanomolar affinities. Cryo-EM shows AlbA as an autoinhibited dimer in which a reciprocal arm closes the NTD end of the partner's tunnel, restricting PBD binding to the CTD while the DNA binding domains (DBDs) are mobile. Albicidin binding is incompatible with this arrangement, whereas PBDs, as CTD plugs, do not perturb the native equilibrium between autoinhibited and promoter-competent states. We visualized the latter by cryo-EM in the RNAP-DNA-AlbA complex. By blocking albicidin-mediated enhancement in vitro, PBDs act as resistance-breaking partners for albicidin and a route to overcoming albA-dependent resistance to oligoarylamide antibiotics.

Recruitment of transcription factors (TFs) to non-cognate DNA sequences is widely observed, but the underlying mechanisms remain poorly understood. Here, we show that, within the nucleosome, Sox2 can bind an Oct4 motif, interact with the Oct4 POU-specific domain, and displace the Oct4 homeodomain. We also observe Sox2 binding at multiple near-canonical and non-canonical DNA sites, with these interactions promoted by a second Sox2 molecule or by nearby nucleosome-bound Oct4. The TF-TF interactions can bridge across DNA gyres, bringing together binding sites that are distant in linear DNA but spatially proximal on the nucleosome. We further find that Sox2 can engage across nucleosomal DNA gyres through loop regions and/or intrinsically disordered segments, providing an additional mode of chromatin binding. Together, our structural and biochemical data reveal how TF-TF interactions change DNA recognition on the nucleosome, enabling binding to non-cognate sequences and expanding the repertoire of pioneer factor interactions on chromatin.

APOBEC3G (A3G), a restriction factor, is a barrier for zoonosis and targeted for degradation by the lentiviral protein Vif. Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2 arose from transmissions of lentiviruses from Old World monkeys to hominid primates. While HIV-1 Vif needed to adapt to specific residues in the arms race interface with A3G, HIV-2 Vif has greater tolerance for diversity there. Here, our cryo-EM structure of HIV-2 Vif and human A3G allows us to explore the mechanism underlying HIV-2 Vif broad specificity. While HIV-2 Vif does bind the arms race interface on A3G, HIV-2 Vif encodes tryptophans with low backbone stability to engage another interface on A3G. We propose a model where flexible loops in HIV-2 Vif undergo conformational selection to recognize diverse A3Gs, whereas the equivalent loops in HIV-1 Vif are preorganized to bind hA3G.

Centromere identity is specified epigenetically by nucleosomes containing the histone H3 variant CENP-A, yet centromeres are almost invariably embedded within repetitive DNA, suggesting that underlying DNA sequences contribute to the organization of centromeric chromatin. In Drosophila melanogaster, CENP-A primarily occupies retroelement-rich islands flanked by simple satellite DNA, but their functional contribution remains unclear. Here, we delete the ~50-kb island Giglio from centromere 3 while preserving the flanking satellites and residual CENP-A chromatin. Despite removal of the DNA sequence associated with most centromeric CENP-A, centromere identity and overall CENP-A levels are preserved at the native locus, indicating that the island itself is not required for centromere identity. Instead, Giglio deletion increases variability in CENP-A chromatin organization, producing highly heterogeneous domain sizes and preferential redistribution of the CENP-A domain into the adjacent dodeca satellite. Together, these findings demonstrate that Giglio is dispensable for centromere identity but constrains the size and position of the CENP-A domain, establishing centromeric islands as key determinants of CENP-A domain architecture.

Cilia are highly conserved organelles that use hundreds of unique proteins to drive extracellular motility and serve as signaling hubs for eukaryotic cells. The Ciliogenesis and Planar Polarity Effector (CPLANE) protein complex controls basal body docking during early ciliogenesis and recruits the intraflagellar transport (IFT) proteins to the base of growing cilia. Leveraging recent advantages in deep learning neural networks and high-performance computing, we predict the structure of JBTS17, the largest CPLANE subunit, and the tertiary structure of the complete CPLANE complex, revealing direct contacts between JBTS17 and three other CPLANE members. We then used this model for an in silico protein-protein interaction screen that identified several novel JBTS17 interactors. Experiments in Xenopus confirmed a functional link in vivo between Jbts17 and the Spinocerebellar ataxia-associated protein TTBK2. The data provide new insights into mechanisms of CPLANE function during ciliogenesis and demonstrate the power of modeling protein complexes for the discovery of physical and functional protein interactions in cell homeostasis and disease.

Structural variants (SVs) are a major source of human genetic diversity, yet their contribution to three-dimensional (3D) genome organization remains difficult to resolve using a standard reference genome. Here, we integrate haplotype-resolved chromatin interaction maps across 354 human and 12 nonhuman ape haplotypes with matched genome assemblies and long-read transcriptomes to determine how inherited SVs influence chromatin organization. We observe significant improvements in contact mapping, with 13.2% of cis Hi-C contacts showing >25 kbp changes in inferred genomic separation and an additional 2.8% of valid contact pairs recovered when using donor-specific assemblies instead of standard references. We show that haplotype-specific chromatin differences are highly selective rather than pervasive, concentrating within 1,790 recurrent contact difference hotspots associated with a restricted subset of SVs. We identified 2,495 chromatin-structure-associated SVs that are enriched in regulatory genomic contexts and are linked to expression variation. Notably, more than 90% of these SVs are derived in the human lineage, consistent with recent remodeling of regulatory architecture at the population level. These findings highlight the importance of incorporating haplotype diversity from pangenomes into studies of chromatin organization and provide a framework for understanding how inherited genomic differences are linked to functional consequences across populations and evolution.

Vascular smooth muscle KATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular KATP is regulated by Mg-nucleotides, but the underlying structural mechanism has remained elusive. Here, we determined cryoEM structures of these channels in the presence of MgATP and MgADP. Two key structures captured, one showing the SUR2B-nucleotide binding domains (NBDs) separated and one showing the SUR2B-NBDs dimerized, reveal conformation-specific organization of intrinsically disordered regions (IDRs) found in both Kir6.1 and SUR2B. In the NBD-separated conformation, the Kir6.1-N terminal IDR (KNt) sits within the central cleft of the ABC-core of SUR2B. In the NBD-dimerized conformation, KNt is excluded from the central cleft and instead forms contacts with an ED domain comprising 15 consecutive glutamate and aspartate residues within a SUR2B IDR, the N1-T2 linker connecting NBD1 (N1) to transmembrane domain 2 (T2). Moreover, within the N1-T2 linker a regulatory helix seen between the two NBDs in the NBD-separated conformation moves to outside the dimerized NBDs, interacting with the C-terminal residues unique to SUR2B, in the NBD-dimerized conformation. MD simulations further reveal that transient but frequent interactions mediated by the IDRs may facilitate Mg-nucleotide dependent conformational switch in vascular KATP channels.