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In the dividing eukaryotic cell, the spindle assembly checkpoint (SAC) ensures that each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex (APC/C), the E3 ubiquitin ligase responsible for initiating chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), which inhibits the APC/C and delays chromosome segregation. By cryo-electron microscopy, here we determine the near-atomic resolution structure of a human APC/C–MCC complex (APC/C MCC ). Degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit responsible for substrate interactions. BubR1 also obstructs binding of the initiating E2 enzyme UbcH10 to repress APC/C ubiquitination activity. Conformational variability of the complex enables UbcH10 association, and structural analysis shows how the Cdc20 subunit intrinsic to the MCC (Cdc20 MCC ) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced. A high-resolution structure of a complex between the anaphase-promoting complex (APC/C) and the mitotic checkpoint complex (MCC) reveals how MCC interacts with and represses APC/C by obstructing substrate recognition and suppressing E3 ligase activity. Basis of mitotic regulation The spindle assembly checkpoint (SAC) is a surveillance mechanism that detects incorrect chromatid kinetochore attachments and delays chromosome segregation by generating a 'wait anaphase' signal. It is activated via the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex (APC/C), a multimeric E3 ligase. Here, David Barford and colleagues use cryo-electron microscopy to determine near-atomic resolution structures of the APC/C–MCC complex. The structures reveal how MCC interacts with and represses APC/C by obstructing substrate recognition and suppressing E3 ligase activity.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins 1 , 2 . Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1 3 . Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO1 4 . The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae . Studies of five cryo-electron microscopy structures reveal the composition and conformational progression in the final maturation events of human 40S ribosomal subunit assembly.

Amyloid fibrils can form biologically relevant functional assemblies. The RIP homotypic interaction motifs (RHIMs) in receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) orchestrate the formation of amyloid-like fibrils essential for propagating cell death signals. While the structures of human RIPK3 (hRIPK3) homomeric fibrils and RIPK1-RIPK3 heteromeric fibrils have been elucidated, the atomic structure of human RIPK1 (hRIPK1) homomeric fibrils has remained elusive. We present a high-resolution structure of hRIPK1 RHIM-mediated amyloid fibrils, determined using an integrative approach combining cryoprobe-detected solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy. The fibrils adopt an N-shaped fold consisting of three β-sheets stabilized by hydrophobic interactions and hydrogen bonding. A key hydrogen bond between N545 and G542 closes the β2-β3 loop, resulting in denser side-chain packing compared to hRIPK3 homomeric fibrils. These findings provide structural insights into how hRIPK1 homomeric fibrils nucleate hRIPK3 recruitment and fibrillization during necroptosis, offering broader perspectives on the molecular principles governing RHIM-mediated amyloid assembly and functional amyloids. This study reveals the atomic structure of RIPK1 fibrils, a type of physiological amyloid that helps control inflammation and regulated cell death in human cells.

The ataxia telangiectasia–mutated and Rad3-related (ATR) kinase is a master regulator of DNA damage response and replication stress in humans, but the mechanism of its activation remains unclear. ATR acts together with its partner ATRIP. Using cryo–electron microscopy, we determined the structure of intact Mec1-Ddc2 (the yeast homolog of ATR-ATRIP), which is poised for catalysis, at a resolution of 3.9 angstroms. Mec1-Ddc2 forms a dimer of heterodimers through the PRD and FAT domains of Mec1 and the coiled-coil domain of Ddc2. The PRD and Bridge domains in Mec1 constitute critical regulatory sites. The activation loop of Mec1 is inhibited by the PRD, revealing an allosteric mechanism of kinase activation. Our study clarifies the architecture of ATR-ATRIP and provides a structural framework for the understanding of ATR regulation.

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose “open” or “closed” position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria. The bacterial stressosome is a large nanomachine and a key inducer of stress response. Here, the authors present the cryo-EM structure of the stressosome from the bacterial pathogen Listeria monocytogenes at 3.38 Å resolution and discuss its activation mechanism.

Haspin, an atypical serine/threonine protein kinase, is a potential target for cancer therapy. 5-iodotubercidin (5-iTU), an adenosine derivative, has been identified as a potent Haspin inhibitor in vitro. In this paper, quantum chemical calculations and molecular dynamics (MD) simulations were employed to identify and quantitatively confirm the presence of halogen bonding (XB), specifically halogen∙∙∙π (aromatic) interaction between halogenated tubercidin ligands with Haspin. Consistent with previous theoretical finding, the site specificity of the XB binding over the ortho-carbon is identified in all cases. A systematic increase of the interaction energy down Group 17, based on both quantum chemical and MD results, supports the important role of halogen bonding in this series of inhibitors. The observed trend is consistent with the experimental observation of the trend of activity within the halogenated tubercidin ligands (F < Cl < Br < I). Furthermore, non-covalent interaction (NCI) plots show that cooperative non-covalent interactions, namely, hydrogen and halogen bonds, contribute to the binding of tubercidin ligands toward Haspin. The understanding of the role of halogen bonding interaction in the ligand–protein complexes may shed light on rational design of potent ligands in the future.

A commonly used strategy by microorganisms to survive multiple stresses involves a signal transduction cascade that increases the expression of stress-responsive genes. Stress signals can be integrated by a multiprotein signaling hub that responds to various signals to effect a single outcome. We obtained a medium-resolution cryo-electron microscopy reconstruction of the 1.8-megadalton \"stressosome\" from Bacillus subtilis. Fitting known crystal structures of components into this reconstruction gave a pseudoatomic structure, which had a virus capsid-like core with sensory extensions. We suggest that the different sensory extensions respond to different signals, whereas the conserved domains in the core integrate the varied signals. The architecture of the stressosome provides the potential for cooperativity, suggesting that the response could be tuned dependent on the magnitude of chemophysical insult.

Cardiac conduction disease (CCD), which causes altered electrical impulse propagation in the heart, is a life-threatening condition with high morbidity and mortality. It exhibits genetic and clinical heterogeneity with diverse pathomechanisms, but in most cases, it disrupts the synchronous activity of impulse-generating nodes and impulse-conduction underlying the normal heartbeat. In this study, we investigated a consanguineous Pakistani family comprised of four patients with CCD. We applied whole exome sequencing (WES) and co-segregation analysis, which identified a novel homozygous missense mutation (c.1531T>C;(p.Ser511Pro)) in the highly conserved kinase domain of the cardiac troponin I-interacting kinase (TNNI3K) encoding gene. The behaviors of mutant and native TNNI3K were compared by performing all-atom long-term molecular dynamics simulations, which revealed changes at the protein surface and in the hydrogen bond network. Furthermore, intra and intermolecular interaction analyses revealed that p.Ser511Pro causes structural variation in the ATP-binding pocket and the homodimer interface. These findings suggest p.Ser511Pro to be a pathogenic variant. Our study provides insights into how the variant perturbs the TNNI3K structure-function relationship, leading to a disease state. This is the first report of a recessive mutation in TNNI3K and the first mutation in this gene identified in the Pakistani population.

The catalytic subunit of the DNA‐dependent protein kinase (DNA‐PKcs) is essential for the repair of double‐stranded DNA breaks (DSBs) in non‐ homologous end joining (NHEJ) and during V(D)J recombination. DNA‐PKcs binds single‐ and double‐stranded DNA in vitro , and in vivo the Ku heterodimer probably helps recruit it to DSBs with high affinity. Once loaded onto DNA, DNA‐PKcs acts as a scaffold for other repair factors to generate a multiprotein complex that brings the two DNA ends together. Human DNA‐PKcs has been analysed by electron microscopy in the absence and presence of double‐stranded DNA, and the three‐dimensional reconstruction of DNA‐bound DNA‐PKcs displays large conformational changes when compared with the unbound protein. DNA‐PKcs seems to use a palm‐like domain to clip onto the DNA, and this new conformation correlates with the activation of the kinase. We suggest that the observed domain movements might help the binding and maintenance of DNA‐PKcs' interaction with DNA at the sites of damage, and that these conformational changes activate the kinase.

The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 heterodimer and an ≈ 460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and protein--nucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein--nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.