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Modern small-angle scattering (SAS) experiments with X-rays or neutrons provide a comprehensive, resolution-limited observation of the thermodynamic state. However, methods for evaluating mass and validating SAS-based models and resolution have been inadequate. Here we define the volume of correlation, V c , a SAS invariant derived from the scattered intensities that is specific to the structural state of the particle, but independent of concentration and the requirements of a compact, folded particle. We show that V c defines a ratio, Q R , that determines the molecular mass of proteins or RNA ranging from 10 to 1,000 kilodaltons. Furthermore, we propose a statistically robust method for assessing model-data agreements ( χ 2 free ) akin to cross-validation. Our approach prevents over-fitting of the SAS data and can be used with a newly defined metric, R SAS , for quantitative evaluation of resolution. Together, these metrics ( V c , Q R , χ 2 free and R SAS ) provide analytical tools for unbiased and accurate macromolecular structural characterizations in solution. Small-angle scattering of X-rays or neutrons is more readily applied to macromolecular complexes than is X-ray crystallography, and is particularly useful for protein complexes with high flexibility; here new quantitative metrics are presented that will allow solution-derived structures to be validated and assessed for mass, resolution and accuracy. New macromolecular structure solutions Small-angle scattering (SAS) of X-rays or neutrons is more readily applied to macromolecular complexes than is X-ray crystallography, and is particularly useful for proteins and complexes with high flexibility. John Tainer and Robert Rambo have developed a new series of quantitative metrics that allow the accuracy of such solution-derived structures to be validated. This refinement strengthens the intrinsic capabilities of SAS for high-throughput analyses and should expand its application to studying flexible macromolecules and nanoparticles in solution.

Although pyroptosis is critical for macrophages against pathogen infection, its role and mechanism in cancer cells remains unclear. PD-L1 has been detected in the nucleus, with unknown function. Here we show that PD-L1 switches TNFα-induced apoptosis to pyroptosis in cancer cells, resulting in tumour necrosis. Under hypoxia, p-Stat3 physically interacts with PD-L1 and facilitates its nuclear translocation, enhancing the transcription of the gasdermin C (GSDMC) gene. GSDMC is specifically cleaved by caspase-8 with TNFα treatment, generating a GSDMC N-terminal domain that forms pores on the cell membrane and induces pyroptosis. Nuclear PD-L1, caspase-8 and GSDMC are required for macrophage-derived TNFα-induced tumour necrosis in vivo. Moreover, high expression of GSDMC correlates with poor survival. Antibiotic chemotherapy drugs induce pyroptosis in breast cancer. These findings identify a non-immune checkpoint function of PD-L1 and provide an unexpected concept that GSDMC/caspase-8 mediates a non-canonical pyroptosis pathway in cancer cells, causing tumour necrosis.Hou et al. show that following hypoxia PD-L1 translocates into the nucleus to enhance transcription of GSDMC, which is then cleaved and activated by caspase-8 to cause pyroptosis in cancer cells.

Transcription preinitiation complexes (PICs) are vital assemblies whose function underlies the expression of protein-encoding genes. Cryo-EM advances have begun to uncover their structural organization. Nevertheless, functional analyses are hindered by incompletely modeled regions. Here we integrate all available cryo-EM data to build a practically complete human PIC structural model. This enables simulations that reveal the assembly’s global motions, define PIC partitioning into dynamic communities and delineate how structural modules function together to remodel DNA. We identify key TFIIE–p62 interactions that link core-PIC to TFIIH. p62 rigging interlaces p34, p44 and XPD while capping the DNA-binding and ATP-binding sites of XPD. PIC kinks and locks substrate DNA, creating negative supercoiling within the Pol II cleft to facilitate promoter opening. Mapping disease mutations associated with xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome onto defined communities reveals clustering into three mechanistic classes that affect TFIIH helicase functions, protein interactions and interface dynamics.A structural model of the human RNA polymerase II preinitiation complex based on high-resolution cryo-EM data provides mechanistic insights into the consequences of human disease mutations.

The DNA damage response (DDR) is an organized network of multiple interwoven components evolved to repair damaged DNA and maintain genome fidelity. Conceptually the DDR includes damage sensors, transducer kinases, and effectors to maintain genomic stability and accurate transmission of genetic information. We have recently gained a substantially improved molecular and mechanistic understanding of how DDR components are interconnected to inflammatory and immune responses to stress. DDR shapes both innate and adaptive immune pathways: (i) in the context of innate immunity, DDR components mainly enhance cytosolic DNA sensing and its downstream STimulator of INterferon Genes (STING)-dependent signaling; (ii) in the context of adaptive immunity, the DDR is needed for the assembly and diversification of antigen receptor genes that is requisite for T and B lymphocyte development. Imbalances between DNA damage and repair impair tissue homeostasis and lead to replication and transcription stress, mutation accumulation, and even cell death. These impacts from DDR defects can then drive tumorigenesis, secretion of inflammatory cytokines, and aberrant immune responses. Yet, DDR deficiency or inhibition can also directly enhance innate immune responses. Furthermore, DDR defects plus the higher mutation load in tumor cells synergistically produce primarily tumor-specific neoantigens, which are powerfully targeted in cancer immunotherapy by employing immune checkpoint inhibitors to amplify immune responses. Thus, elucidating DDR-immune response interplay may provide critical connections for harnessing immunomodulatory effects plus targeted inhibition to improve efficacy of radiation and chemotherapies, of immune checkpoint blockade, and of combined therapeutic strategies.

In this study, 83 proteins containing helix–loop–helix–loop repeats were designed—with sequences unrelated to known repeat proteins—and experimentally characterized; 43 solution X-ray scattering spectra and 15 structures of the designed proteins show that these non-natural repeat proteins have a broad range of curvatures and that their overall structures are in close agreement with design models. Tandem repeat proteins by design Repeat proteins are composed of multiple tandem copies of a modular structure unit and are widespread in nature, playing critical roles in molecular recognition, signalling, and other essential biological processes. In natural repeat proteins, the interactions between adjacent units define the shape and curvature of the overall structure. Two papers published in this issue of Nature describe the design of geometrically unconstrained, open tandem repeat arrays. David Baker and colleagues used computational protein design to generate a series of proteins containing repeats of a simple 'helix-loop-helix-loop' structural motif. Data from 43 proteins with solution X-ray scattering spectra, and 15 structures of the designed proteins, show that these non-natural repeat proteins have a broad range of curvatures and that their overall structures are in close agreement with design models. Philip Bradley and colleagues used computational protein design to synthesize a series of alpha-solenoid/toroid structures that have various radii and different sized 'holes'. The authors solved X-ray crystal structures of four of the designed proteins and determined that their overall structures are in close agreement with the design models. A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit 1 are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes 2 . Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications 3 , 4 , 5 . Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix–loop–helix–loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

Transcription-coupled repair (TCR) is a vital nucleotide excision repair sub-pathway that removes DNA lesions from actively transcribed DNA strands. Binding of CSB to lesion-stalled RNA Polymerase II (Pol II) initiates TCR by triggering the recruitment of downstream repair factors. Yet it remains unknown how transcription factor IIH (TFIIH) is recruited to the intact TCR complex. Combining existing structural data with AlphaFold predictions, we build an integrative model of the initial TFIIH-bound TCR complex. We show how TFIIH can be first recruited in an open repair-inhibited conformation, which requires subsequent CAK module removal and conformational closure to process damaged DNA. In our model, CSB, CSA, UVSSA, elongation factor 1 (ELOF1), and specific Pol II and UVSSA-bound ubiquitin moieties come together to provide interaction interfaces needed for TFIIH recruitment. STK19 acts as a linchpin of the assembly, orienting the incoming TFIIH and bridging Pol II to core TCR factors and DNA. Molecular simulations of the TCR-associated CRL4 CSA ubiquitin ligase complex unveil the interplay of segmental DDB1 flexibility, continuous Cullin4A flexibility, and the key role of ELOF1 for Pol II ubiquitination that enables TCR. Collectively, these findings elucidate the coordinated assembly of repair proteins in early TCR. Transcription-coupled repair (TCR) removes DNA lesions from actively transcribed strands. Here, the authors unveil the structural basis for coordinated TFIIH recruitment to the TCR machinery and the role of ubiquitin modifications in directing the response toward DNA repair or proteasomal degradation.

DNA polymerase theta (POLθ) is synthetic lethal with Homologous Recombination (HR) deficiency and thus a candidate target for HR-deficient cancers. Through high-throughput small molecule screens we identified the antibiotic Novobiocin (NVB) as a specific POLθ inhibitor that selectively kills HR-deficient tumor cells and . NVB directly binds to the POLθ ATPase domain, inhibits its ATPase activity, and phenocopies POLθ depletion. NVB kills HR-deficient breast and ovarian tumors in GEMM, xenograft and PDX models. Increased POLθ levels predict NVB sensitivity, and BRCA-deficient tumor cells with acquired resistance to PARP inhibitors (PARPi) are sensitive to NVB and Mechanistically, NVB-mediated cell death in PARPi-resistant cells arises from increased double-strand break end resection, leading to accumulation of single-strand DNA intermediates and non-functional RAD51 foci. Our results demonstrate that NVB may be useful alone or in combination with PARPi in treating HR-deficient tumors, including those with acquired PARPi resistance. (151/150).

Interleukin (IL)-33 is an important member of the IL-1 family that has pleiotropic activities in innate and adaptive immune responses in host defense and disease. It signals through its ligand-binding primary receptor ST2 and IL-1 receptor accessory protein (IL-1RAcP), both of which are members of the IL-1 receptor family. To clarify the interaction of IL-33 with its receptors, we determined the crystal structure of IL-33 in complex with the ectodomain of ST2 at a resolution of 3.27 Å. Coupled with structure-based mutagenesis and binding assay, the structural results define the molecular mechanism by which ST2 specifically recognizes IL-33. Structural comparison with other ligand–receptor complexes in the IL-1 family indicates that surface-charge complementarity is critical in determining ligand-binding specificity of IL-1 primary receptors. Combined crystallography and small-angle X-ray–scattering studies reveal that ST2 possesses hinge flexibility between the D3 domain and D1D2 module, whereas IL-1RAcP exhibits a rigid conformation in the unbound state in solution. The molecular flexibility of ST2 provides structural insights into domain-level conformational change of IL-1 primary receptors upon ligand binding, and the rigidity of IL-1RAcP explains its inability to bind ligands directly. The solution architecture of IL-33–ST2–IL-1RAcP complex from small-angle X-ray–scattering analysis resembles IL-1β–IL-1RII–IL-1RAcP and IL-1β–IL-1RI–IL-1RAcP crystal structures. The collective results confer IL-33 structure–function relationships, supporting and extending a general model for ligand–receptor assembly and activation in the IL-1 family.

Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adapter for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS–STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination. GRB2 is known for its role in Receptor Tyrosine Kinase and RAS signaling. Here the authors unveil a GRB2 function and mechanism for DNA replication fork protection. GRB2 alleviates oncogenic replication stress, and in doing so, averts cancer immune destruction by inhibiting cGAS/STING and pro-inflammatory cytokine production.

Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival. PARG catalyzes the removal of poly(ADP-ribose) (PAR) from target proteins and executes critical functions in the DNA damage response. Here the authors provide structural and biological insight with small molecule PARG inhibitors and show that PARG inhibition sensitizes cells to ionizing radiation and kills cancer cells through replication fork defects.