Nature Structural & Molecular Biology

Cation-proton antiporters (CPAs) are vital for the maintenance of ionic homeostasis and normal physiology among diverse cell types. Despite recent insights into K+/H+ exchange transporters, the diversity in their structural organization and regulatory mechanisms of K+-specific CPAs are minimally understood. Here, we explore the architecture of an E. coli CPA1 K+/H+ antiporter, YcgO and its inhibition by the unphosphorylated form of PtsN, the terminal protein of a regulatory phosphorelay, using cryoEM structures at 3.4 [A] and 3.2 [A] resolution, respectively. Homodimeric YcgO bound to K+ ions in the occluded conformation, harbors additional linked cytosolic domains, RCK and CorC, to regulate the movement of the transport helices within the YcgO dimer. These domains are the sites of interaction and efflux inhibition by unphosphorylated PtsN, which interacts with the CorC domains with high affinity and allosterically augments inhibitory interactions of CorC with transport helices of YcgO. Inhibition is relieved leading to constitutive activation, upon disrupting the CorC-transport conduit interface. This study illuminates the structural basis of K+ efflux mediated through regulation of a K+/H+ antiporter in E. coli and related prokaryotes via a metabolic network involving a regulatory phosphorelay.

CRISPR technologies based on nuclease-deactivated Cas9 (dCas9) rely on programmable DNA binding rather than DNA cleavage, yet the intrinsic DNA-recognition properties that govern optimal guide RNA (gRNA) performance remain poorly understood. Existing approaches either measure genomic occupancy in cells or infer dCas9 behavior from cleavage-based Cas9 datasets, despite DNA binding being substantially more permissive than DNA cleavage. Here we introduce TANGO (Targeted Array-based Nucleic acid-Guided Occupancy), a high-density DNA-array platform that quantitatively profiles intrinsic dCas9:gRNA binding across tens of thousands of DNA targets in a cell-free system. TANGO captures established features of dCas9 target recognition, while providing substantially greater sensitivity than prior assays. Comparison with ChIP-seq data demonstrates that intrinsic DNA-binding specificity is a major driver of genomic occupancy and reveals that chromatin accessibility modulates the intrinsic binding affinity required for dCas9 recruitment. Across CRISPRi/a guides, TANGO identifies multiple independent biochemical determinants of guide performance--including on-target affinity, mismatch tolerance, and ribonucleoprotein assembly--and flags problematic and highly promiscuous guides overlooked by current specificity metrics. Unexpectedly, some guides retain substantial guide-directed DNA binding even in the absence of a protospacer-adjacent motif (PAM), revealing an additional dimension of dCas9 specificity. Together, these results establish intrinsic DNA recognition as a quantitative and experimentally accessible determinant of dCas9 function, providing a framework for improving guide selection and enhancing the precision of CRISPR technologies.

Targeted protein degradation (TPD) has emerged as an increasingly powerful approach for therapeutic development and biological discovery. TPD compounds including proteolysis-targeting chimeras (PROTACs), molecular glues, and tag-targeting protein degraders (tTPD) enable rapid, selective and reversible degradation of proteins through recruitment of the ubiquitin-proteasome system (UPS). However, genome-wide CRISPR screens performed with targeted protein degraders are frequently dominated by resistance mechanisms that disrupt degrader activity, including loss of recruited E3 ligase components and broader UPS regulators. The strong selective advantage conferred by these perturbations can obscure less penetrant, biological genetic interactions that operate downstream of target degradation. To overcome this limitation, through iterative genome-wide screening and manual curation, we developed a TPD-compatible CRISPR knockout library that retains near-genome-scale coverage while excluding a focused set of genes recurrently associated with degrader failure. Across multiple degrader screens, this library reduced the dominance of UPS-associated resistance mechanisms and improved the detection and prioritization of genetic interactions linked to target biology. Using the RBM39 molecular glue degrader indisulam as a model, we identified ZMAT2 loss as a resistance mechanism that preserves RBM39 degradation but attenuates the transcriptional and splicing consequences of target depletion. Together, our work establishes a TPD-compatible CRISPR screening framework that improves the biological resolution of degrader resistance screens and facilitates the discovery of genetic dependencies operating downstream of targeted protein degradation.

Cellular self-organisation counteracts entropy at the expenditure of energy. In case of the first level of nuclear DNA organisation, this relates to regular nucleosome arrays and interspaced nucleosome-depleted regions (NDRs), for example at promoters or replication origins1-5. The organisation of nucleosomes as building blocks of chromatin is orchestrated by the collective activities of ATP-dependent chromatin remodellers6-9. Yet, how remodellers achieve positional specificity, in particular regarding the promoter-proximal +1 nucleosomes, remains unclear. Here, we show that the S. cerevisiae chromatin remodeller INO80 unexpectedly distinguishes DNA sequence asymmetry within the +1 nucleosome of the SWH1 gene through distinct inhibited and active nucleosome-binding modes. In structural and biochemical analyses of INO80 on nucleosomes with the endogenous sequence, we identified an inhibited binding mode where the entire INO80 remodelling unit flipped on the +1 nucleosome. INO80 adopted this remodelling-incompetent binding mode when facing the promoter, but a remodelling-competent mode when facing the gene body. This directional read-out of intra-nucleosomal DNA sequence asymmetry, together with extranucleosomal NDR sequence features, prevented nucleosome sliding into the NDR while permitting array formation over the gene. Our work shows how DNA features contribute to ATP-dependent self-organisation of promoter chromatin by INO80.

Factor XI (FXI) is a key coagulation protease of the intrinsic pathway of blood coagulation and an emerging antithrombotic target. However, the structural transition from zymogen to active Factor XIa (FXIa) has remained poorly understood. Using cryo-EM, we demonstrate that FXI activation results in a global reorganization of the homodimer, extending beyond the activation loop to include a significant reorientation of the catalytic domain (CD) relative to the apple-domain (AD) platform. The CD displays pronounced conformational heterogeneity; we identify three distinct conformers, suggesting that FXIa exists as a dynamic ensemble rather than a single rigid state. MD analysis indicates that activation disrupts the inter-CD allosteric communication present in the zymogen, thereby facilitating this flexibility. CD plasticity allows for the dynamic exposure of the A3 exosite, facilitating the binding of Factor IX. Comparison with plasma kallikrein (PKa) suggests that such structural flexibility may be a shared feature of apple-domain-containing contact-system proteases. Our results reveal that FXIa functions as a dynamic ensemble, providing a structural framework for understanding substrate recognition and identifying novel, non-catalytic sites for the development of specific FXIa inhibitors.

Genes encoding novel protein sequences are a ubiquitous feature of genomes. They fuel molecular and cellular evolutionary innovations and frequently contribute to species-specific characteristics. We are now unravelling the processes by which they originate, including de novo from noncoding sequences and through extreme divergence, yet how much and what types of novel proteins evolve through each process is still unclear Does the mechanism of origination shape the structural and functional potential of the resulting proteins? Here, we conducted a broad computational investigation of genetic and protein novelty at the scale of the entire subphylum of Saccharomycotina yeasts. We detected more than 5,000 robust de novo genes across 332 species and compared them to more than 10,000 novel genes resulting from extreme sequence divergence, revealing two distinct modes of evolution of novelty. A remarkable 40% of de novo proteins are predicted to localize to mitochondria compared to only 15% of divergent, with the latter also being substantially longer and more disordered. A detailed analysis of conservatively predicted tertiary structures of novel proteins shows that "invention" of novel folds can happen through both processes but is more likely to occur de novo. We also illustrate cases of evolutionary "re-invention" of existing protein folds from non-coding sequences. Our work deepens our understanding of the origins and importance of novel proteins opening new directions for further structural and functional characterization.

Human PARP9 harbours two tandem macro domains, MD1 and MD2, with distinct roles in ADP-ribosylation signaling. Whereas MD1 is a MacroD-type de-MARylase ("eraser"), MD2 functions as a MacroH2A-like "reader" lacking detectable hydrolase activity. How two domains so similar in sequence and fold achieve such divergent functionality has remained unclear. Our de-MARylation assays confirmed this division of labor, even though crystal structures revealed nearly identical /{beta}/ folds and similar binding pockets, and solution NMR shows broadly comparable dynamics. A remarkable distinction, however, emerges from isothermal titration calorimetry, showing that both domains bind free ADP-ribose with comparable affinity (KD = 5.4 and 8.4 M), but through markedly different thermodynamics: MD1 binding is enthalpy-driven and offset by a larger entropic penalty, whereas MD2 binds with weaker enthalpy and a smaller entropic cost. Our ADPr-bound crystal structures rationalized this showing that the distal ribose is positioned differently in the two pockets, connecting to a glycine-rich catalytic loop and a conserved aromatic residue present in active macro domains like MD1, but altered in MD2, while the catalytic asparagine itself is structurally conserved (Asn140/Asn339). Moreover, we show that a single amino acid substitution in MD2 leads to detectable RNA de-MARylation activity without altering its fold. Together, our findings show that an eraser-versus-reader distinction between the macrodomains in PARP9 is encoded in the dynamics of ligand binding and the exact position the distal ribose, revealing an unexpected catalytic plasticity relevant to PARP9s roles in immunity and cancer.

Cholesterol is a key component of cellular membranes and is critical for brain function, particularly axon myelination. Among the 48 human ATP-binding cassette (ABC) transporters, ABCA2 exhibits the highest expression in the brain and is involved in cholesterol metabolism, primarily in oligodendrocytes. Notably, ABCA2 has been associated with myelin sheath integrity and maintenance, as well as Alzheimer's disease. Here, we report cryo-EM structures of human ABCA2 that reveal critical endogenous lipid-binding sites unique to ABCA2. Our five distinct conformations include a previously uncharacterized intermediate between the closed and apo states of ABCA subfamily transporters. Most importantly, we elucidated the cholesterol transport mechanism of ABCA2, which involves novel interdependent rotations of the exocytoplasmic domains (ECDs) and regulatory domains (RDs). Our structural findings provide a new perspective on ABCA transporter function and highlight the role of ABCA2 in facilitating efficient cholesterol recycling and transport in the brain.

Respiratory complex I, a central enzyme in cellular metabolism, converts the free energy of NADH oxidation into a transmembrane proton-motive force to drive ATP synthesis, but the molecular mechanisms by which it couples redox catalysis to vectorial proton translocation remain unresolved. Here, we present high-resolution cryo-EM structures of complex I from Bos taurus captured under conditions designed to change the protonation states of residues in the membrane domain. Our structures reveal conformational rearrangements at key pathway junctions that reconfigure proton-transfer connections. In ND5, helical rearrangements switch the connectivity of histidine-248 between proton-uptake and proton-output pathways. In ND4, rotameric changes of histidine-220 alternately enable proton uptake or lateral proton transfer along the membrane domain. Combined with molecular simulations, our structures define gating mechanisms that impose directionality on proton transfer reactions and provide a framework for proton-coupled energy transduction in complex I.

Subtelomeres are imperfect repeats adjacent to telomeres that are transcriptionally repressed by heterochromatin. Although essential for genome integrity, their repetitive nature has thwarted dissection of local heterochromatin assembly and maintenance mechanisms. By engineering Schizosaccharomyces pombe strains carrying fluorescent reporters at a single subtelomere, we uncovered distinct subdomains. These subdomains have different silencing requirements: Telomere-proximal regions rely on canonical shelterin- or RNAi-dependent nucleation pathways, whereas telomere-distal regions involve nucleosome remodelers, histone chaperones, and boundary-associated factors. Subdomains also exhibit discrete epigenetic states emerging both at homologous loci on different chromosome arms and along the same subtelomeric sequence. We document these epigenetic states using multi-generational live imaging and targeted perturbations. These analyses show that subtelomeric subdomains display position-specific, clonally variable silencing across a spectrum from robust to fragile epigenetic states. Interestingly, structural variants, common in subtelomeric sequences across eukaryotes, dictate local epigenetic stability. These findings reveal that subtelomeres, long recognized for their sequence variability, form a dynamic mosaic of coexisting epigenetic domains. This implies a wide range of gene-repressive regulatory logic at the chromosome ends, from environmental responsiveness to silencing stability.

Inner mitochondrial membrane proteins must be sufficiently hydrophilic to withstand aqueous exposure during translation and transit to the mitochondria. Meanwhile, their transmembrane segments must be sufficiently hydrophobic to stably embed in the lipid membrane. We hypothesized that sequence-level adaptations evolved to balance these constraints. Here, we integrate structure-informed evolutionary analyses of mitochondrial proteins with atomistic simulations and cell-based experiments to identify aliphatic-to-threonine substitutions (ATS) as a potential solution to these constraints. With high statistical confidence, this transmembrane segment-specific adaptation is recurrently and convergently observed throughout mitochondrial evolution. Conformational analyses show that threonine interacts with both water and the transmembrane helix backbone, thereby lowering hydrophobicity without destabilizing secondary structure. In the extremely hydrophobic ATP6 protein, reverting threonines to aliphatic residues disrupts mitochondrial targeting, while introducing threonines into a poorly targeted variant improves its mitochondrial localization. These findings have implications for mitochondrial genome evolution, the rational design of membrane proteins, and potentially mitochondrial gene therapy.

Diet has repeatedly shaped human genomes, yet adaptation of protein digestion remains poorly understood. Using 1,348 haplotype-resolved assemblies, we reconstructed the structural evolution of the human pepsinogen A (PGA) locus and found a west-to-east increase in copy-number across Eurasia that tracked regional reliance on plant-derived protein. Modern and ancient genomes further revealed a recent selective sweep in the East but signatures of balancing selection in the West. We traced this divergence primarily to expansion of PGA34A, the most proteolytically active paralog in vitro, and showed that recurrent nonallelic homologous recombination continually generated structural diversity in this locus. Independent PGA expansions were also enriched in plant-dominant mammals. These findings link paralog-specific dosage variation in protein digestion to dietary adaptation across human populations and diverse mammalian lineages.

Deciphering the regulatory consequences of sequence divergence across human evolution is essential to understanding the molecular basis of human-specific traits and disease. Although millions of derived alleles distinguish humans from great apes, only a small fraction are likely to influence human-specific traits. Previous studies have focused on regions of elevated sequence divergence, assuming that rapid evolution reflects functional adaptation, yet individual high-impact regulatory mutations evade such scans. Here, we apply sequence-to-function deep learning to predict chromatin accessibility across modern human, archaic hominin, and great ape personalized genomes, identifying lineage-specific cis-regulatory elements (linCREs) across diverse cellular contexts. Compared to conserved elements, linCREs are shorter, less pleiotropic, less conserved, and enriched in neurodevelopmental pathways. Many linCREs occur in regions with limited sequence divergence that acceleration-based approaches would overlook. We validate lineage-specific enhancer activity through luciferase reporter assays and demonstrate that a single motif-generating derived allele nominated by model interpretability tools drives a hominin-specific neurodevelopmental enhancer.

Genomic DNA is packaged into chromatin through a fundamental repeating unit known as the nucleosome core particle. Chromatinized DNA is under constant assault from endogenous and exogenous sources of damage, which must be effectively repaired to preserve genome stability. Single-strand breaks (SSBs) with chemically heterogeneous DNA ends are one of the most prevalent forms of genomic DNA damage. These SSBs must be enzymatically processed prior to downstream gap-filling DNA synthesis and/or nick ligation during single-strand break repair (SSBR). Polynucleotide kinase phosphatase (PNKP) is a multifunctional end-processing enzyme that possesses two catalytic activities important for converting non-ligatable SSBs into ligatable SSBs. To date, a mechanistic description for how PNKP processes non-ligatable SSBs in the context of chromatin to initiate SSBR remains undefined. Here, we utilize a combination of biochemical assays and cryogenic electron microscopy (cryo-EM) to define the structural basis of end processing in the nucleosome by PNKP. Cryo-EM structures of PNKP engaged with non-ligatable SSBs at three unique positions within the nucleosome reveal that PNKP locally deforms nucleosomal DNA to reposition the SSBs into the kinase and phosphatase active sites, providing a structural basis for the efficient processing of SSBs throughout the nucleosome. Additional cryo-EM structures reveal the PNKP FHA domain also engages the nucleosome acidic patch during non-ligatable SSB recognition, which accelerates the processing of non-ligatable SSBs in the nucleosome. Together, these findings provide important mechanistic insight into the initial end processing step of chromatin-based SSBR.

The molecular composition of the mitochondrial permeability transition pore (mPTP) remains contested, and several efficacious mPTP inhibitors act through undefined, cyclophilin D (CypD)-independent targets. Using two structurally distinct chemotypes of optimised, brain-penetrant mPTP inhibitors as chemical probes, we applied affinity-based chemoproteomics to identify the mitochondrial NOD-like receptor NLRX1 as their shared target. Both chemotypes bind NLRX1, and binding potency across a compound series tracks mPTP-inhibitory activity. Using CRISPR-Cas9-edited human cells and Nlrx1-/- mouse tissues, we show that NLRX1 is required for normal calcium-induced mPTP opening: its loss raises the calcium threshold for pore opening and its overexpression lowers it, independently of CypD. NLRX1 associates with postulated mPTP components, including ATP synthase and the adenine nucleotide translocase, in a compound-sensitive manner, and sustains mitochondrial protein homeostasis over longer timescales. The lead compound, GSK900, is orally bioavailable, brain-penetrant, and active in an mPTP-sensitive neurological injury model. These findings, converging with recent genetic studies, establish NLRX1 as an essential, CypD-independent regulator of mitochondrial permeability transition and provide brain-penetrant chemical tools to interrogate this biology.

Initiator-methionine excision is among the earliest protein modifications, yet its relationship to assembly geometry is unknown. Burial of the mature first residue was measured across 7,246 deposited human biological assemblies (22,291 chain-level observations; 1,191 proteins). Among 1,143 analyzable proteins, termini in MetAP-permissive penultimate-residue sequence classes were less often interface-engaged than termini in MetAP-nonpermissive classes (37.4% versus 47.4%; adjusted odds ratio 0.65, p = 7.2e-4). Curated processing annotations did not show a corresponding burial difference, and correlated residue properties preclude attributing the sequence-class association specifically to iMet removal. The analysis identified 264 interface-engaged MetAP-permissive candidates concentrated in cellular machines. In a fully recomputed conformer scan of deeply buried proteasome positions, modeled methionine accommodation was less favorable than at observed-methionine controls (median overlap -0.30 versus -1.12 angstrom, p = 0.0049), although most scoreable sites permitted a nonoverlapping placement. The census therefore reveals a graded structural constraint - not universal steric failure - and prioritizes complexes in which altered packing, assembly kinetics, lipidation or N-terminal methylation can be tested.

Pioneer transcription factors direct cell differentiation by remodeling closed chromatin to deploy new enhancers. It remains unclear how the chromatin context shapes their action. During pituitary development, the pioneer PAX7 opens thousands of enhancers and specifies the melanotrope cell fate. We report that PAX7 acts in both the A (active) and B (inactive) genomic compartments, engaging two distinct closed states: H1-enriched chromatin in A, and H3K9me2- and lamin-rich chromatin in B. Whereas A-compartment enhancers open in a single, cell-division-independent step, B-compartment opening requires cell division and triggers domain-wide lamin dissociation coupled with B-to-A compartment shift. The two modes involve distinct biological outputs: A-compartment action modulates broadly expressed genes, whereas B-compartment action drives de novo expression of cell-type-specific developmental genes central to melanotrope identity.

ATP-dependent chromatin remodeling enzymes play central roles in genome regulation by mobilizing nucleosomes. All remodelers except for one, position their core ATPase at an internal nucleosome location, superhelical location (SHL) 2, and translocate DNA from this site to alter nucleosome conformation. The exception, the remodeler INO80, positions its core ATPase, Ino80, near the DNA exit/entry site, SHL -6. This major architectural difference has raised the question of whether INO80 acts by a fundamentally different mechanism from other remodelers. Here, using cryogenic electron microscopy we capture a new conformation of INO80 that is substantially enriched upon ATP hydrolysis and has Ino80 positioned at an internal nucleosomal location. Comprehensive conformational landscape analysis further uncovers an ATP hydrolysis dependent continuum of additional INO80 conformational states on a nucleosome that were not previously detected. Our studies provide compelling evidence to support a model where INO80 initially engages nucleosomes near SHL -6, followed by a dramatic ATP hydrolysis dependent ~180{degrees} reorientation around the nucleosome to place its Ino80 near SHL -2 from where DNA is translocated. INO80s unique reorientation has broad implications for understanding how ATP-dependent steps that precede nucleosome mobilization can increase the fidelity of remodeling by being responsive to nucleosomal cues.

Large-scale structural variants (SVs) are major drivers of genome evolution and disease susceptibility, shaping lineage-specific gene content, genome architecture, and phenotypic diversity. Yet, their systematic detection across divergent species has been hindered by complex rearrangements and reference bias, limiting our understanding of their evolutionary impact. Here we present LGvar, a divergence-aware tool for identifying SVs in population-scale and cross-species genome assemblies, enabling up to a 7-fold increase in the detection of large deletions, insertions, inversions and complex rearrangements ([&ge;]10 kbp). Application of LGvar uncovers 112 previously undetected large SVs overlooked by conventional methods and quantifies the effect of evolutionary divergence on SV discovery across primate euchromatic genomes, revealing an approximately 30% overall decline in detection sensitivity with increasing phylogenetic distance. Mapping SVs from comparable syntenic regions onto the primate phylogeny shows that large deletions ([&ge;]10 kbp) accumulate approximately two-fold faster than insertions and eight-fold faster than inversions, in contrast to the dynamics of smaller SVs (<10 kbp). Integrating structurally divergent regions with the synteny region analysis reveals over 606 previously unreported gene gains and losses in great apes and refined the totals to 134 human-lineage-specific and 51 great-ape-lineage-specific protein coding genes. These results suggest that large SVs follow distinct evolutionary trajectories, with potential selective constraints shaping genome structure. Our study establishes a generalizable strategy for cross-species SV discovery and provides a high-resolution view of how large SVs contribute to genome evolution in primates.

The actin cytoskeleton rapidly reorganizes in response to intracellular calcium signals, driving cellular morphogenesis and wound healing. Among actin regulators, the formin INF2 uniquely mediates the "Calcium-mediated Actin Reset" (CaAR) reaction, orchestrating transient and global actin remodeling upon calcium influx. Excessive INF2 activity is linked to kidney and neuronal diseases, underscoring the need for its tight control. Combining live cell imaging with single molecule tracking, biochemistry and structural analysis we discover that INF2 activity is tightly controlled by two interlinked mechanisms: canonical intramolecular autoinhibition and binding of the INF2 N-terminus to the side of actin filaments. Side-binding limits actin elongation and supports re-establishment of autoinhibition. Disruption of this negative feedback prolongs INF2 activity, affecting plasma membrane organization and repair as well as transcriptional control. Our findings uncover a novel product-inhibition mechanism that limits INF2 function and offer important insight into disease mechanisms linked to actin dysregulation.