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Abstract: Background: IgE is the central driver of allergic responses. Prior studies have defined the conformation of the IgE Fc fragment bound to the FcεRIα ectodomain and the dynamic properties of the IgE Fc. It remains unknown, how these prior studies translate to the complex of a full antibody including the Fab arms with the receptor. Methods: For structural analysis, crystallography, cryo-EM and negative stain EM (ns-EM) were combined. IgE variants were analyzed by mediator release and CD23 binding assays. Results: An ensemble of 10 cryo-EM structures of the full-size IgE FcεRIα complex was obtained revealing that the receptor bound IgE adopts a pronounced T-like conformation. Either Fab arm may rotate up to 40°. Two additional conformations with different arrangements of the Fab arms were captured in ns-EM. The introduction of additional flexibility into the Fab-Fc hinge does not compromise the biological activity of IgE, suggesting that the observed conformations of the IgE Fab-Fc hinge exhibit equivalent biological function. Comparison of the full IgE receptor complex with recent cryo-EM structures of the intact receptor reveals that FcεRI conformations differ markedly by the orientation of the ectodomain. Hence, our ensemble of IgE FcεRIα structures including the Fab arms enabled critical evaluation of FcεRI conformations. Conclusion: Our data reveal the architecture of a full-size IgE antibody bound to its receptor and a new layer of dynamics in FcεRIα bound IgE on top of the well-established IgE Fc conformations. Development of novel anti-IgE therapeutics may take into account these properties of FcεRIα bound IgE.

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Abstract: Cyclin-dependent kinases (CDKs) are prototypical regulators of the cell cycle. The CDK-activating kinase (CAK) acts as a master regulator of CDK activity by catalyzing the activating phosphorylation of CDKs on a conserved threonine residue within the regulatory T-loop. However, structural data illuminating the mechanism by which the CAK recognizes and activates CDKs have remained elusive. Here, we determine high-resolution structures of the CAK in complex with CDK2 and CDK2-cyclin A2 by cryogenic electron microscopy. Our structures reveal a T-loop–independent kinase-kinase interface with contributions from both kinase lobes. Computational analysis and structures of CAK in complex with CDK1-cyclin B1 and CDK11 indicate that these structures represent the general architecture of CAK-CDK complexes. These results advance our mechanistic understanding of cell cycle regulation and kinase signaling cascades.

Open Access

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Abstract: CDK7 has emerged as a cancer target because of its pivotal roles in cell cycle progression and transcription. Several CDK7 inhibitors (CDK7i) are now in clinical evaluation. Identifying patients most likely to respond to treatment and early detection of tumour evolution towards resistance are necessary for optimal implementation of cancer therapies. Continuous culturing of prostate cancer cells with Samuraciclib, a non-covalent ATP-competitive CDK7i, led to outgrowth of resistant cells. These were characterised by the acquisition of a single base change in the CDK7 gene, Asp97 to Asn (D97N). Mutant cells were resistant to other non-covalent CDK7i but remained sensitive to covalent CDK7i. Cryo-EM structure and kinase ligand affinity determinations revealed reduced affinity of the CDK7-D97N mutant for non-covalent CDK7i. Remarkably, Asp97 is absolutely conserved in human CDKs, inferring its importance for the activities of all CDKs. Consistent with this, mutation of the homologous residue in CDK12 (D819N) or CDK4 (D99N) promoted resistance to drugs that inhibit these CDKs. Our findings reveal a general mechanism for acquired resistance with obvious implications for patients treated with CDK inhibitors.

Open Access

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Abstract: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is central to global CO2 fixation. In eukaryotic algae, its catalytic efficiency is enhanced through the pyrenoid - a protein-dense organelle within the chloroplast that concentrates CO2. Although Rubisco structure has been extensively studied in vitro, its native structure, dynamics and interactions within the pyrenoid remain elusive. Here, we present the native Rubisco structure inside the green alga Chlamydomonas reinhardtii determined by cryo-electron tomography and subtomogram averaging of cryo-focused ion beam milled cells. Multiple structural subsets of Rubisco are identified, stochastically distributed throughout the pyrenoid. While Rubisco adopts an active conformation in the best-resolved map, comparison among the subsets reveals significant local variations at the active site, at the large subunit dimer interfaces, and at binding protein contact regions. These findings offer a comprehensive understanding of the structure, dynamics, and functional organization of native Rubisco within the pyrenoid, providing valuable insights into its critical role in CO2 fixation.

Open Access

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Abstract: Lentiviruses, such as HIV-1, infect non-dividing cells by traversing the nuclear pore complex (NPC); however, the detailed molecular processes remain unclear. Here we reconstituted functional HIV-1 nuclear import using permeabilized T cells and isolated HIV-1 cores, which significantly increases import events, and developed an integrated three-dimensional cryo-correlative workflow to specifically target and image 1,489 native HIV-1 cores at 4 distinct nuclear import stages using cryo-electron tomography. We found HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability. The NPC acts as a selective filter, preferentially importing smaller cores, while expanding and deforming to accommodate their passage. Brittle mutant cores fail to enter the NPC, while CPSF6-binding-deficient cores enter but stall within the NPC, leading to impaired nuclear import. This study uncovers the interplay between the HIV-1 core and the NPC and provides a framework to dissect HIV-1 nuclear import and downstream events, such as uncoating and integration.