Caleigh M. Azumaya

Kinetochores are essential macromolecular complexes anchoring chromosomes to the mitotic spindle, ensuring faithful cell division. Despite their critical role, the structural organization of kinetochores across diverse species remains poorly understood. We present the inner kinetochore constitutive centromere-associated network (CCAN) structure of the silkmoth Bombyx mori , an insect that lacks the canonical centromere-specifying histone variant CENP-A and exhibits chromosome-wide centromeric activity (holocentric). The B. mori CCAN incorporates four previously uncharacterized centromeric subunit proteins that are structurally related to the Dam1/DASH complex but function in scaffolding the inner kinetochore rather than in microtubule binding. Similar to the yeast and human systems, the B. mori CCAN also entraps DNA within its central closed chamber. However, unlike these systems, the B. mori CCAN can also assemble in vitro into a self-contained head-to-head dimer via atypical histone-fold protein dimerization. On the basis of our findings, we propose that the holocentric organization may emerge from the modular arrangement of discrete kinetochore units.

C-X-C motif chemokine receptor 3 (CXCR3) is essential for immune cell functions and pivotal in T helper 1 cell infiltration in autoimmune and chronic inflammatory diseases and in tumor proliferation and metastasis, but the mechanisms by which the endogenous ligands CXCL9, CXCL10, and CXCL11 differentially recognize and activate CXCR3 are not fully understood. Here, we present cryo–electron microscopy structures of all three chemokine-CXCR3-G _i complexes, complemented by cell binding studies and functional mutagenesis data. We systematically compare the pharmacological and interaction profiles of CXCL9, CXCL10, and CXCL11 to rationalize their varying efficacies and potencies and to reveal the critical role of the membrane-distal CXCR3 N terminus in ligand binding and signaling. Using chimeric chemokines and molecular dynamics, we reveal the signaling plasticity of chemokine ligands and signaling determinants. Together, these insights enable us to propose a multimodal binding and activation framework that explains CXCR3 chemokine ligand multispecificity and signaling versatility and offer tools to interrogate and modulate CXCR3 biology.

Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase that regulates the phosphorylation of almost all cellular processes, including cell division and proliferation^1,2. PP2A forms heterotrimeric holoenzyme complexes comprising a catalytic subunit (PP2Ac), a scaffolding subunit (PP2Aa) and variable B regulatory subunits that exert precise control over enzyme substrate specificity and prevent indiscriminate dephosphorylation of phosphoproteins³. However, the mechanisms that control the activity of uncomplexed catalytic subunits have remained relatively unclear. Here we find that the E3 ligase SKP1-CUL1-F-box (SCF) complex containing F-box other protein 42 (FBXO42, also known as JFK; hereafter, SCF^FBXO42) degrades holoenzyme-free PP2Ac in a complex with the coiled-coil protein CCDC6 to maintain cancer cell fitness. The cryo-electron microscopy structure of the FBXO42-CCDC6-PP2Ac assembly reveals a pseudosymmetric architecture in which CCDC6 forms a central dimeric template that recruits multiple copies of PP2Ac and creates a substrate for FBXO42. Both the quaternary structure of this CCDC6-PP2Ac heterodimer and the post-translationally methylated tail of PP2Ac are recognized by FBXO42 for ubiquitination. The multivalent structure facilitated by CCDC6 enables the assembly of multiple degradation complexes along a single coiled coil, leading to the turnover of free phosphatases and downregulation of catalytic activity. Together, our findings define a mechanism for PP2A control through the ubiquitin-proteosome system and establish a paradigm for cullin-RING ligase-substrate interactions.

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of inflammatory responses in asthma, allergy and other diseases. Here we report an anti-β-tryptase antibody with a superior mechanism of action compared to others since it not only inhibits tetrameric β-tryptase, but also completely inhibits monomeric β-tryptase activity. The antibody binds to an exosite that causes tetramer dissociation as either an IgG or Fab and, in addition, allosterically alters the substrate binding cleft on monomers, thus preventing substrate binding and proteolysis. We solve the cryoEM structure of the complex, generate biochemical data and engineer point mutations to elucidate the allosteric path of inhibition. This ultimately reveals a single Asp to Gly mutation in CDR-L3 that only slightly impacts binding affinity, but completely eliminates inhibitory activity. Finally, we improve antibody inhibitory potency up to 4.7-fold by structure-based design creating new charge-charge interactions. This antibody may have enhanced efficacy and potential to assess the relevance of β-tryptase, including monomers, in biological and clinical settings.

The repair of DNA double-strand breaks by homologous recombination (HR) is essential for genomic integrity, and its dysregulation is a hallmark of cancer¹. Central to HR is the RAD51 recombinase, whose assembly into a nucleoprotein filament is governed by five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3)². Mutations in any of these proteins predispose individuals to multiple cancers or genetic disorders^3-6. These paralogs are thought to form two functionally separate complexes, BCDX2 (RAD51B-C-D-XRCC2) and CX3 (RAD51C-XRCC3), that act independently at different stages of HR^7-11. Here, we demonstrate that all five paralogs can assemble into a single, ATP-dependent BCDX2-CX3-RAD51 supercomplex. The architecture of this assembly bound to single-stranded DNA (ssDNA) reveals a contiguous filament where the CX3 module stacks atop BCDX2, creating a protofilament template for RAD51 filament formation. We further identify a novel, RAD51B-independent DX2-CX3 complex (RAD51D-XRCC2-RAD51C-XRCC3) functioning as a stable RAD51 anchor on ssDNA, and we capture it in multiple states, including capping RAD51 filament segment. These distinct assemblies are differentially regulated by ATPase activity, defining a dynamic BCDX2-CX3 “loader” and a stable DX2-CX3 “anchor” that provide functional modularity to the HR machinery. This work provides a unifying mechanism for human RAD51 paralog function and delivers an atomic blueprint for interpreting disease-causing mutations.

Integrins are heterodimeric receptors important for cell adhesion and signaling. Integrin α5β1 is a key mediator of angiogenesis and its dysregulation is associated with tumor progression and metastasis. Despite numerous efforts, α5β1-targeting therapeutics have been unsuccessful due to poor efficacy and off-target effects. A contributing factor is our limited understanding of how integrin conformation influences interactions with therapeutics. Using cell-based functional assays, patient derived xenografts, biophysics, and electron microscopy, we shed light on these relationships by characterizing two anti-α5β1 antibodies, BIIG2 and MINT1526A. We show that both antibodies bind α5β1 with nanomolar affinity, reduce angiogenesis in vitro , and bind overlapping epitopes that block fibronectin binding. However, using cryoEM, we reveal that while BIIG2 binding doesn’t alter the conformational states, MINT1526A restricts α5β1’s range of flexibility. These insights can guide which aspects to prioritize and improve the design of future integrin-targeted therapeutics.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

Ferritin is a multivalent, self-assembling protein scaffold found in most human cell types, in addition to being present in invertebrates, higher plants, fungi, and bacteria, that offers an attractive alternative to polymer-based drug delivery systems (DDS). In this study, the utility of the ferritin cage as a DDS was demonstrated within the context of T cell agonism for tumor killing. Members of the tumor necrosis factor receptor superfamily (TNFRSF) are attractive targets for the development of anticancer therapeutics. These receptors are endogenously activated by trimeric ligands that occur in transmembrane or soluble forms, and oligomerization and cell-surface anchoring have been shown to be essential aspects of the targeted agonism of this receptor class. Here, we demonstrated that the ferritin cage could be easily tailored for multivalent display of anti-OX40 antibody fragments on its surface and determined that these arrays are capable of pathway activation through cell-surface clustering. Together, these results confirm the utility, versatility, and developability of ferritin as a DDS.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

Nanobodies that neutralize: Monoclonal antibodies that bind to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) show therapeutic promise but must be produced in mammalian cells and need to be delivered intravenously. By contrast, single-domain antibodies called nanobodies can be produced in bacteria or yeast, and their stability may enable aerosol delivery. Two papers now report nanobodies that bind tightly to spike and efficiently neutralize SARS-CoV-2 in cells. Schoof et al. screened a yeast surface display of synthetic nanobodies and Xiang et al. screened anti-spike nanobodies produced by a llama. Both groups identified highly potent nanobodies that lock the spike protein in an inactive conformation. Multivalent constructs of selected nanobodies achieved even more potent neutralization. Science , this issue p. 1473 , p. 1479