Nature Structural & Molecular Biology

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.

The pyruvate dehydrogenase complex (PDHc)1 links glycolysis to the Krebs cycle by catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2, a process essential for life2,3. PDHc is formed by structural proteins (E3-binding protein, E3BP)4-7, enzymatic subunits (E1, E2, E3)4,6, and mobile lipoyl domains (LDs), the latter shuttling intermediates across active sites6,8. Although numerous details regarding pyruvate oxidation steps have been elucidated9, the precise organization of the entire PDHc remains unknown due to its large size and dynamic heterogeneity. Here, we employ in silico, in vitro, and in situ methods to propose a multi-scale model of PDHc that includes approximately one million atoms and to visualize multiple conformational states. This model reveals a [~]40-50 nm nested shell structure, formed by flexible linkers that spatially coordinate the E1 and E3 enzyme complexes around the E2-E3BP core scaffold. This structure acts as a molecular sieve, selectively guiding lipoyl arms while maintaining enzyme positioning with sub-nm precision. During catalysis, the nested shell structure expands and adopts a mechanically reinforced state comparable in magnitude to viral assemblies10. Our findings provide structural context for the textbook "link reaction"11, building on decades of biochemical knowledge, are transferable to functional aspects of other -ketoacid dehydrogenase complexes, and, ultimately, expand our understanding of primary metabolism as a whole. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/724543v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1615612org.highwire.dtl.DTLVardef@159bc8dorg.highwire.dtl.DTLVardef@69f8c1org.highwire.dtl.DTLVardef@14a83a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

The anteroposterior collinear expression of Hox genes is a hallmark of animal development that underpins the diversification of body plans1 and life cycles2. However, the origin and drivers of this coordinated expression remain elusive: while vertebrates rely on complex cluster-wide Hox gene regulation3-8, insects define gene-specific, sub-cluster regulatory domains9-11. Here, we discover a new mode of Hox gene regulation in segmented worms (Annelida). By combining chromatin conformation data with histone modifications profiling in Owenia fusiformis, we show that a large distal enhancer forms developmentally regulated, long-range contacts across the Hox cluster, and its activation coincides with the consolidation of a cluster-wide topologically associating domain (TAD), loss of Polycomb-mediated repression, and Hox gene transcription. This chromatin structure also occurs in the annelids Dimorphilus gyrociliatus and Capitella teleta, the latter showing additional subTAD structures that correlate with Hoxs temporal collinearity12. Moreover, related phyla with intact, organised Hox clusters and spatial collinearity, such as nemerteans and chitons, show annelid-like chromatin organisations, whereas phyla with disorganised13 Hox clusters do not. Coordinated Hox gene regulation from a "global control region" is thus ancestral to Lophotrochozoa, indicating that complex regulatory logics based on cluster-wide, long-range chromatin interactions with distal enhancers evolved convergently in vertebrates and spiralians.

Cell-free expression using Leishmania tarentolae lysates allows rapid expression of eukaryotic proteins directly from DNA templates. We develop a pipeline that combines cell-free expression system with cryogenic fluorescence microscopy that we term CC-FLEXCET (Correlative Cell-Free Leishmania EXpression and Cryo-Electron Tomography), to target and visualize expressed protein complexes by cryo-electron tomography at high resolution. We demonstrate the utility of this method by structurally characterising the filaments of the full-length apoptosis-associated speck like protein containing CARD (ASC) protein. Cell-free expression of ASC results in a polymeric structure characteristic of its cellular speck assembly. Sub-tomogram averaging allows us to resolve both the pyrin domain (PYD) to medium resolution, and show, for the first time, the arrangement of the flexibly linked caspase recruitment domain (CARD). Finally, we observed an interaction between the ASC filament and the L. tarentolae ribosome. Using template matching and quantitative approaches, we characterise this interaction and determine that there is a random structural association between the filament and the ribosome, with 57% of ribosomes oriented with the LSU toward the ASC polymer. CC-FLEXCET facilitates structural analysis of macromolecules and protein-lipid assemblies without need of purification, providing a pipeline from DNA template to protein expression to cryo-tilt series acquisition, within a single day.

Structural heterogeneity in biomolecules, arising from both compositional and conformational variability, limits resolution and interpretability of cryo-electron tomography (cryo-ET). Here, we present OPUS-ET, a deep learning framework that resolves multiscale heterogeneity throughout the cryo-ET workflow. OPUS-ET combines a composition decoder that captures compositional differences with a conformation decoder that models large-scale motions, thereby providing a hierarchical representation of structural heterogeneity. Starting from noisy template-matching candidates with templates of varying similarity or quality, OPUS-ET efficiently enriches target particle populations and delivers sub-nanometer in situ reconstructions in a single round. It leads to improved resolutions by up to 4.5 [A] over expert annotations or existing deep-learning approaches in four benchmark systems, and reveals continuous conformational landscapes capturing F-F flexible coupling in mitochondrial ATP synthase and tRNA-translocation intermediates in eukaryotic and bacterial ribosomes. Together, these results establish OPUS-ET as a powerful computational tool for linking particle purification, high-resolution reconstruction, and analysis of structural heterogeneity in cryo-ET, with demonstrated robustness to template quality, initial pose noise, and clustering parameters.

Ribosome composition can vary through differences in associated proteins, post-transcriptional and post-translational modifications. Such heterogeneity enables ribosomes to respond to environmental1 or pathological2,3 conditions, and modulate localized translation4. A long-standing observation has also been the differential expression of variant ribosomal RNA (rRNA) alleles across developmental5-7 or cellular states8-14. Yet how exchanging the catalytic ribosome core could regulate translational outcomes remains unknown. Here, we report the functional characterization of a genomically-encoded, divergent rRNA that serves as a dominant-negative repressor of translation during host-to-vector transmission in the human malaria parasite. This allele only encodes for large subunit rRNAs, lacks ITS2 splicing, yet retains conserved rRNA modification and folding patterns alongside vast expansion segments. The resulting large subunit engages mRNA at translation start sites but appears to elongate inefficiently, likely due to divergences in the peptidyl transferase center obstructing the exit tunnel. Through its precisely timed transcription immediately after transmission, this rRNA represses mRNAs that were highly translated in the human, facilitating the transition of the translational program for mosquito-stage development. Our data identify a repressive ribosome population whose antagonistic function is encoded by an independently evolved, variant rRNA allele, defining the conceptual foundation for an additional layer of inherent translational regulation.

Heterochromatin Protein 1 (HP1) is a fundamental component of constitutive heterochromatin, forming subnuclear condensates whose regulation and function remain poorly understood. Here, we present an image-based CRISPR screen targeting nuclear factors that identifies splicing as a pivotal pathway regulating HP1 condensates. We discovered that unspliced intronic RNA modulates HP1 condensates by interacting co-transcriptionally with HP1. By modulating the intron content, RNA processing restricts HP1-RNA interactions at chromatin, thus enabling heterochromatin organization. Disruption of HP1 condensates due to enhanced interactions with unspliced RNA leads to loss of heterochromatin and the activation of stress response protective genes. We propose that RNA is a central component of heterochromatin that modulates HP1 condensates, and that RNA processing enzymes act as a surveillance mechanism for condensates by dynamically regulating the network of multi-valent interactions between RNA and chromatin factors. This model underscores the crosstalk between chromatin organization, transcription, and RNA processing, potentially governing broader nuclear functions.

Microtubule nucleation is commonly viewed as a {gamma}-tubulin ring complex ({gamma}-TuRC)-templated process 1-3, yet many differentiated cells build extensive non-centrosomal microtubule arrays of unclear origin 4-6. Although spontaneous tubulin nucleation has long been observed in vitro as a {gamma}-TuRC-independent nucleation process 7-10, its mechanism and cellular relevance have remained unclear. Here we show that CAMSAP2, a microtubule minus-end-binding protein, links spontaneous nucleation to non-centrosomal microtubule organization. Cryo-electron microscopy (cryo-EM), high-speed atomic force microscopy (hsAFM), and molecular dynamics simulations reveal that CAMSAP2 lowers the nucleation barrier by straightening tubulin oligomers and promoting lateral protofilament interactions that drive sheet formation and closure into microtubules. hsAFM captures stepwise nucleation and early growth, revealing that tubulin rings can serve as productive intermediates rather than dead-end depolymerization products. CAMSAP2 further self-organizes through liquid-liquid phase separation (LLPS) to concentrate tubulin and assembly intermediates, thereby promoting efficient microtubule formation at non-centrosomal microtubule-organizing centers. In HeLa cells, dispersed CAMSAP2 condensates act as {gamma}-TuRC-independent microtubule-organizing centers alongside centrosomal asters, supporting a model in which spontaneous nucleation contributes to non-centrosomal microtubule biogenesis.

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.

Transcription initiation by RNA polymerase II (Pol II) occurs next to a +1 nucleosome that is positioned downstream of the transcription start site (TSS). The +1 nucleosome has been shown to influence the pre-initiation complex (PIC) assembly and Pol II pausing, but it is unclear whether and how it functions in transcription initiation and the transition to elongation. Here, we investigate the transcription initiation-elongation transition in vitro using DNA templates containing a +1 nucleosome, and we present cryo-EM structures of five intermediate states. First, after PIC assembly, the +1 nucleosome evicts TFIID from the PIC upon binding of ATP to TFIIH. Second, after DNA opening, the +1 nucleosome stimulates TFIIH translocase activity and initial RNA synthesis. Finally, after DNA bubble rewinding, the +1 nucleosome removes TFIIH from the early elongation complex for promoter escape. Our findings show that the +1 nucleosome not only acts as a passive border for PIC assembly and a passive barrier for Pol II pausing, but rather has active functions during the initiation-elongation transition of transcription.

Summary paragraphWhen cells enter S phase, bidirectional DNA replication is initiated through the kinase-regulated recruitment of three activators (Cdc45, GINS and Pol epsilon) to a duplex DNA-loaded double hexamer of MCM ATPases. Together these proteins form two CMGE helicases that establish divergent replication forks as they become separated1. To understand CMGE biogenesis, we reconstituted the pre-Initiation Complex with purified yeast proteins. The cryo-EM structure shows a set of firing factors caught in the act of assembling two symmetric CMGEs. We show how stepwise complex formation reshapes MCM in preparation for DNA opening and we explain how ATP promotes firing-factor ejection and CMGE maturation. While we find that Sld2 promotes GINS recruitment to MCM as expected, it also aids efficient separation of the CMGE dimer, and it is essential for lagging strand ejection from MCM. These findings have direct implications for our understanding of the metazoan Sld2 ortholog, RECQL4, pointing to a replication-fork establishment mechanism conserved across eukaryotes.

Chromosome conformation capture (3C)-derived methods have become an indispensable tool in the study of gene regulation. The three-dimensional contacts probed by 3C methods depend strongly on the properties of the enzyme used to fragment chromatin prior to proximity-driven ligation. Micrococcal nuclease (MNase), used in Micro-C, increases resolution at the expense of low ligation efficiency and the need for extensive enzyme titration. To overcome these limitations, we engineered a highly active, TEV protease-activatable caspase-activated DNase (CAD) to enable an efficient, low-sequence-bias, and high-resolution proximity ligation assay we call CAD-C. CAD-C was successful on the first attempt for each human cell line tested and the resulting datasets capture loops, TADs, compartments, and stripes similarly to Micro-C. However, compared to Micro-C and Hi-C, CAD-C shows enhanced sensitivity for promoter-enhancer loops. Leveraging the ligation-competent DNA ends produced by CAD cleavage, we show that CAD-C is compatible with a highly streamlined, repair-free protocol and produces multi-step CADwalks, consecutive ligations between nucleosomal or sub-nucleosomal fragments. With these walks, we probe local chromatin fiber folding contacts, nucleosomal and sub-nucleosomal footprints, and long-range nuclear organization regimes in human cell lines. CAD-C is an efficient, robust chromatin structure assay that can span sub-nucleosomal to chromosomal length scales in a single experiment.

Biogenesis of subcellular structures like centrioles is viewed as a physical transformation wherein elementary constituents form order without preexisting templates. Centrioles grow with precision from a composite scaffold known as the cartwheel, which is thought to self-assemble without templates and disassemble following centriole growth; however, the mechanism governing cartwheel assembly-disassembly dynamics remains obscure. Here, we identify ALMS1, a disease-linked, intrinsically disordered protein (IDP), as an external mediator of cartwheel dynamics that causes a seed for cartwheel--and thus centriole--formation without itself incorporating into the seed structure. The cartwheel seed (CS), characterized as a dense composite of CEP152/CEP63 protein complexes, forms in interphase and adopts a nanoscale, concentric ring from which the cartwheel grows. Upon mitotic entry, CSs recruit ALMS1 while disassembling into constituents associating with ALMS1 in proximity, correlating with cartwheel assembly-disassembly cycles. Hypomorph, disease-linked ALMS1 mutations trigger cartwheel expansion and shedding by its own grown procentriole, in turn forming ectopic centrioles, leading to perpetual reciprocal amplification. Without ALMS1, CS formation fails, negating centriole biogenesis, whereas reintroducing ALMS1 initializes biogenesis anew, creating diverse yet heritable architectures that evolve through selection, instead of generating a single canonical form. These results suggest that centriole biogenesis is grounded on adaptable transformation cues extrinsic to its constituents, propagating via IDP-mediated CS assembly-disassembly cycles, a process we conjecture involves memory.

H2A.Z and canonical H2A adopt nearly identical nucleosomal folds, yet their distinct chromatin functions are not captured by static structural analysis. Using fast magic-angle spinning 1H-detected solid-state NMR, we show that H2A.Z possesses enhanced backbone flexibility in the L1 loop and the 2-L2 region (M2) relative to H2A. Chimeric segment-swapping demonstrates that these dynamic signatures are locally sequence-encoded and functionally transplantable. The inherent mobility of the M2 region promotes nucleosomal DNA-end unwrapping and persists when DNA ends are stabilized by linker histone H1 or opened by SUV420H1, indicating that this mobility is intrinsic rather than a passive consequence of DNA detachment. Chemical shift perturbation mapping and catalytic assays further show that SUV420H1 reads this H2A.Z-specific conformational landscape: the M2 region, together with the H2A.Z DS motif, supports variant-selective methyltransferase activity. These findings establish an axis of sequence-dynamics-accessibility-recognition along which local backbone fluctuations serve as physical determinants of epigenetic enzyme specificity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/731474v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@a50ceforg.highwire.dtl.DTLVardef@1f43af0org.highwire.dtl.DTLVardef@14969b8org.highwire.dtl.DTLVardef@bbbe0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Long-read sequencing has enabled precise measurements of highly repetitive centromeric satellites and their rapid divergence between species1-7. Large satellite arrays emerge from libraries of shorter arrays via stochastic expansions8,9, but understanding the selective pressures constraining such expansions remains a major challenge. Here, using the mouse "major" satellite as a model, we reveal reciprocal functional constraints between increasing satellite copy number and abundance of a conserved architectural protein in female meiosis. We show that HMGA2 (high mobility group AT-hook 2) is enriched at major satellite, and its expression correlates with major satellite copy number: both are high in Mus musculus compared to the closely related Mus spretus. To test functional constraints, we modulated HMGA2 abundance by depletion or overexpression and used a musculus/spretus hybrid to generate oocytes with intermediate HMGA2 expression and major satellite copy number. We find that HMGA2 depletion disrupts major satellite packaging in major satellite-rich musculus but not hybrid oocytes, indicating that increasing copy number requires high HMGA2 expression. Conversely, HMGA2 overexpression disrupts chromosome segregation in major satellite-poor spretus but not hybrid oocytes, indicating that high HMGA2 expression requires expanded major satellite arrays. Based on these results, we propose a co-evolution model in which satellite expansion is constrained by architectural protein abundance, whereas protein abundance is constrained reciprocally by satellite array size.

CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex1,2. Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools3-6. The type I-F Pseudoalteromonas CAST (PseCAST) displays the highest activity in mammalian cells to date7 and has been the subject of extensive directed evolution8, but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation9. Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the PseCAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa PseCAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.

The virulence of emerging Gram-negative pathogens frequently arises from toxins delivered by the type II secretion system1. Cryo-EM single particle analysis and cryo-electron tomography and have defined the outer membrane secretin pore in detail, but the organisation of proteins within the periplasm and inner membrane that form the pilus assembly platform is not well resolved2,3. Here we combine AlphaFold4 models with single particle cryo-EM to define the organisation of the pilus assembly platform. We show that CLM heterotrimers form a continuous link from the cytoplasmic ATPase, across the inner membrane and periplasm, to the base of the secretin channel. AlphaFold models of the inner membrane spanning rotor and cytoplasmic ATPase fit readily within the cryo-EM density. The resolved secretion system exhibits an offset between the inner membrane assembly platform and the outer membrane secretin pore, together with profound asymmetry and an unexpectedly open periplasmic architecture. This architecture provides a route by which large, folded proteins access the secretion channel from the periplasm and suggests that substrate engagement may trigger the final steps in secretion system assembly leading to secretion.

OLD, an abortive immunity protein from prophage P2, consists of an ABC ATPase sensor and a TO-PRIM nuclease effector - a core architecture shared by a large protein family, including components of anti-phage systems Gabija, PARIS, Septu, and Lamassu. OLD was originally identified for its lethality in recB-deficient cells and inhibition of bacteriophage {lambda} infection, but the mechanisms governing its activation have remained elusive. Here, we present the cryo-EM structure of an inactive OLD tetramer and show that destabilization into dimeric form opens the TOPRIM catalytic site, stimulating tRNA cleavage. This activity arrests translation, a phenotype rescued by phage-encoded tRNAs. We demonstrate that OLD activation is not strictly RecBCD-dependent: OLD binds aberrant DNA structures in recB-deficient cells, but activation during infection requires recognition of single-stranded DNA hairpins at the phage replication origin. Collectively, our findings reveal how host and phage DNA processing factors create a complex landscape controlling OLD-mediated immunity.

Lamina-associated domains (LADs) are large genomic regions that interact with the nuclear lamina (NL). Much of the underlying "grammar" governing their positioning at the nuclear periphery remains unclear. LADs are composed of heterochromatin and typically harbor repressed genes, and their association with the NL is generally incompatible with strong transcriptional activity. The extent to which transcription globally shapes chromatin-NL interactions is not fully understood. Here, we combined acute transcription inhibition using Flavopiridol or Triptolide with genome-wide mapping of chromatin-NL contacts. We found that chromatin-NL interactions are rapidly rewired upon transcription inhibition. Changes in chromatin-NL contacts upon transcription shutdown are predictable based on transcriptional activity and the presence of H3K9me3-marked heterochromatin. This rewiring is reversible, as genome-NL interactions quickly return to baseline levels following drug wash-off. Notably, gain and loss of chromatin-NL interactions upon transcription shutdown reflect two distinct tethering mechanisms. Inter-LADs genomic regions (iLADs) enriched in highly active genes and located near stable LADs, which are tethered by Lamin A (LMNA/C), become re-attached to the NL following transcription inhibition. In parallel, H3K9-methylated regions tethered to the nuclear envelope by the Lamin B receptor (LBR) undergo extensive detachment from the NL. Strikingly, LMNA/C and LBR oppositely regulate transcription-sensitive LADs and are required for transcriptional control of chromatin-NL contacts. Together, our findings highlight the plasticity and dynamic nature of chromatin-NL interactions and provide the first evidence that LMNA/C- and LBR-mediated tethering mechanisms exhibit distinct sensitivities to transcription inhibition. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/738400v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1c35abborg.highwire.dtl.DTLVardef@79c483org.highwire.dtl.DTLVardef@547ce8org.highwire.dtl.DTLVardef@d4790e_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LITranscription inhibition alters chromatin-NL contacts rapidly and reversibly C_LIO_LIActive transcription prevents inter-LADs located near LMNA/C-tethered LADs from associating with the nuclear lamina. C_LIO_LILBR-tethered heterochromatin is repositioned away from the NL C_LIO_LITranscription-dependent modulation of chromatin-NL contacts is dependent on LMNA/C and partially on LBR C_LI

Glutamate dehydrogenase (GDH) is a highly regulated key enzyme that catalyzes the reversible oxidative deamination of glutamate to alpha-ketoglutarate, positioning it at a critical hub linking amino acid catabolism to energy production while supplying ammonia for urea synthesis and other nitrogen pathways. Early investigations have shown that bovine GDH (bGDH), which shares 98% sequence identity with its human homolog, assembles into polymeric filaments with altered allosteric responses. Filamentation has only relatively recently been appreciated as a widespread mechanism of enzyme regulation, prompting a reevaluation of these early observations in GDH. Here, we use high resolution cryogenic electron microscopy (cryo EM) to show that bGDH hexamers assemble via reciprocal antenna interactions that oppose the conformational changes associated with GTP inhibition, revealing how filamentation reshapes GDH allostery and with implications for the treatment of human disease.