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DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128–amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.

Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, β-, γ-, δ-, ε-, ρ-, θ- and π-subunits 1 . αβ, α4βδ, α6βδ and α5βγ receptors favour extrasynaptic localization, and mediate an essential persistent (tonic) inhibitory conductance in many regions of the mammalian brain 1 , 2 . Mutations of these receptors in humans are linked to epilepsy and insomnia 3 , 4 . Altered extrasynaptic receptor function is implicated in insomnia, stroke and Angelman and Fragile X syndromes 1 , 5 , and drugs targeting these receptors are used to treat postpartum depression 6 . Tonic GABAergic responses are moderated to avoid excessive suppression of neuronal communication, and can exhibit high sensitivity to Zn 2+ blockade, in contrast to synapse-preferring α1βγ, α2βγ and α3βγ receptor responses 5 , 7 – 12 . Here, to resolve these distinctive features, we determined structures of the predominantly extrasynaptic αβ GABA A receptor class. An inhibited state bound by both the lethal paralysing agent α-cobratoxin 13 and Zn 2+ was used in comparisons with GABA–Zn 2+ and GABA-bound structures. Zn 2+ nullifies the GABA response by non-competitively plugging the extracellular end of the pore to block chloride conductance. In the absence of Zn 2+ , the GABA signalling response initially follows the canonical route until it reaches the pore. In contrast to synaptic GABA A receptors, expansion of the midway pore activation gate is limited and it remains closed, reflecting the intrinsic low efficacy that characterizes the extrasynaptic receptor. Overall, this study explains distinct traits adopted by αβ receptors that adapt them to a role in tonic signalling. Cryo-electron microscopy structures are used to identify mechanisms underlying distinct features of extrasynaptic type A γ-aminobutyric acid receptors.

DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form DNA-dependent protein kinase holoenzyme (DNA-PK) in the process of non-homologous end joining (NHEJ). We present a 2.8-Å-resolution cryo-EM structure of DNA-PKcs, allowing precise amino acid sequence registration in regions uninterpreted in previous 4.3-Å X-ray maps. We also report a cryo-EM structure of DNA-PK at 3.5-Å resolution and reveal a dimer mediated by the Ku80 C terminus. Central to dimer formation is a domain swap of the conserved C-terminal helix of Ku80. Our results suggest a new mechanism for NHEJ utilizing a DNA-PK dimer to bring broken DNA ends together. Furthermore, drug inhibition of NHEJ in combination with chemo- and radiotherapy has proved successful, making these models central to structure-based drug targeting efforts. A new cryo-EM structure of human DNA-PKcs in complex with a Ku70/80 heterodimer and DNA reveals how Ku80–DNA-PKcs interactions create a scaffold to mediate DNA double-strand break repair.

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3′-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3′-cleavage and 3′-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5′ and 3′ tRNA-end processing, methylation and 3′-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3′-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria. Mitochondrial tRNAs are less structurally stable than nuclear tRNAs, and their maturation pathway is unique. Here, the authors reveal how human mitochondrial precursor tRNAs are recognised, processed, methylated and prepared for full functionality in mitochondrial translation.

Turning the HEAT on DNA-PKcs Several members of the phosphatidylinositol-3-OH kinase (PI(3)K) family are involved in the response to DNA double-strand breaks. One of these, DNA-dependent protein kinase (DNA-PK), is comprised of three subunits, with the kinase activity residing in the catalytic subunit, DNA-PKcs. In this study, Tom Blundell and colleagues have solved the structure of human DNA-PKcs, at a resolution sufficient to see the overall folds. The structure reveals that the many HEAT repeats bend the protein into a circular structure. The kinase domain, encoded in the C-terminal domain, sits on one side of the structure. While the overall architecture of the catalytic subunit allows speculation about regions where conformational changes may occur, confirmation of such interactions awaits higher resolution data. If broken chromosomes arising from DNA double-strand breaks are left unrepaired or incorrectly repaired, they can lead to genomic changes that may result in cell death or cancer. DNA-dependent protein kinase (DNA-PK), which comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer Ku70/Ku80, has a major role in the repair of double-strand breaks. The crystal structure of human DNA-PKcs is now presented, in which the overall fold is clearly visible. Broken chromosomes arising from DNA double-strand breaks result from endogenous events such as the production of reactive oxygen species during cellular metabolism, as well as from exogenous sources such as ionizing radiation 1 , 2 , 3 . Left unrepaired or incorrectly repaired they can lead to genomic changes that may result in cell death or cancer. DNA-dependent protein kinase (DNA-PK), a holoenzyme that comprises the DNA-PK catalytic subunit (DNA-PKcs) 4 , 5 and the heterodimer Ku70/Ku80, has a major role in non-homologous end joining—the main pathway in mammals used to repair double-strand breaks 6 , 7 , 8 . DNA-PKcs is a serine/threonine protein kinase comprising a single polypeptide chain of 4,128 amino acids and belonging to the phosphatidylinositol-3-OH kinase (PI(3)K)-related protein family 9 . DNA-PKcs is involved in the sensing and transmission of DNA damage signals to proteins such as p53, setting off events that lead to cell cycle arrest 10 , 11 . It phosphorylates a wide range of substrates in vitro , including Ku70/Ku80, which is translocated along DNA 12 . Here we present the crystal structure of human DNA-PKcs at 6.6 Å resolution, in which the overall fold is clearly visible, to our knowledge, for the first time. The many α-helical HEAT repeats (helix–turn–helix motifs) facilitate bending and allow the polypeptide chain to fold into a hollow circular structure. The carboxy-terminal kinase domain is located on top of this structure, and a small HEAT repeat domain that probably binds DNA is inside. The structure provides a flexible cradle to promote DNA double-strand-break repair.

In nature, sophisticated functional materials are created through hierarchical self-assembly of simple nanoscale motifs 1 , 2 , 3 , 4 . In the laboratory, much progress has been made in the controlled assembly of molecules into one- 5 , 6 , 7 , two- 6 , 8 , 9 and three-dimensional 10 artificial nanostructures, but bridging from the nanoscale to the macroscale to create useful macroscopic materials remains a challenge. Here we show a scalable self-assembly approach to making free-standing films from amyloid protein fibrils. The films were well ordered and highly rigid, with a Young's modulus of up to 5–7 GPa, which is comparable to the highest values for proteinaceous materials found in nature. We show that the self-organizing protein scaffolds can align otherwise unstructured components (such as fluorophores) within the macroscopic films. Multiscale self-assembly that relies on highly specific biomolecular interactions is an attractive path for realizing new multifunctional materials built from the bottom up. Well-ordered and highly rigid macroscopic films can be self-assembled from amyloid protein fibrils.

DNA double-strand breaks (DSBs) are highly deleterious lesions that can trigger cell death or carcinogenesis if unrepaired or misrepaired. In mammals, most DSBs are repaired by non-homologous end joining (NHEJ), which begins when Ku70/80 binds DNA ends and recruits DNA-PKcs to form the DNA-PK holoenzyme. Although recent cryo-EM studies have resolved several NHEJ assemblies, how these factors access DSBs within nucleosomes remains unclear. Here, we present cryo-EM structures of human Ku70/80 and DNA-PK bound to nucleosomes. Ku70/80 binds the DNA end and bends it away from the nucleosome core, while the Ku70 C-terminal SAP domain makes an additional, specific DNA contact. Our DNA-PK–nucleosome structure further reveals the opening of the Ku80 vWA domain, and we show that non-hydrolysable ATP promotes synapsis by stabilising the Ku80-mediated DNA-PK dimer. These structures reveal a model for DSB recognition on nucleosomal DNA and provide insights relevant to targeting NHEJ in cancer therapy. DNA double-strand breaks endanger genome stability. Here, the authors present cryo-EM structures showing how Ku70/80 and DNA-PK bind DNA ends on nucleosomes, offering a mechanistic model for break recognition within chromatin.

Aedes aegypti has evolved to become an efficient vector for arboviruses but the mechanisms of host-pathogen tolerance are unknown. Immunoreceptor Toll and its ligand Spaetzle have undergone duplication which may allow neofunctionalization and adaptation. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A complexes that display transient but specific interactions with Spaetzle1C, forming asymmetric complexes, with only one ligand clearly resolved. Loop structures of Spaetzle1C and Toll5A intercalate, temporarily bridging the receptor C-termini to promote signalling. By contrast unbound receptors form head-to-head homodimers that keep the juxtamembrane regions far apart in an inactive conformation. Interestingly the transcriptional signature of Spaetzle1C differs from other Spaetzle cytokines and controls genes involved in innate immunity, metabolism and tissue regeneration. Taken together our results explain how upregulation of Spaetzle1C in the midgut and Toll5A in the salivary gland shape the concomitant immune response. Aedes aegypti can act as a vector for viral pathogens but the mechanism of viral resistance and evolving host-pathogen tolerance are poorly understood. Here the authors structurally characterise a duplicated pair of interacting Toll immunoreceptors and the cytokine ligand Spaetzle1C and show their dose-dependent function and mechanism of activation.

The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years 1 , 2 . However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABA A receptor homopentamer 3 . Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery. Advances in electron cryo-microscopy hardware allow proteins to be studied at atomic resolution.

Type A γ-aminobutyric acid receptors (GABA A Rs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits 1 , 2 and can be modulated by essential medicines including general anaesthetics and benzodiazepines 3 . Human GABA A R subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABA A Rs assembled from α4, β3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors 4 – 7 . This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABA A R arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data 8 , 9 , we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABA A Rs. The diverse makeup and assembly of subunits augment the structure, physiology and pharmacology of GABA A receptors.