Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from a ...substrate to a competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, thereby inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation, and in rodents it enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by cryo-electron microscopy. Formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and ISRIB promoted this step by cross-bridging a central symmetry interface. Thus, regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB.
The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; ...however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.
The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a guanosine triphosphatase ...that becomes activated by eIF2B, a two-fold symmetric and heterodecameric complex that functions as eIF2's dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from a substrate into an inhibitor of eIF2B. We report cryo-electron microscopy structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B's bipartite catalytic centers to catalyze nucleotide exchange. Phosphorylation refolds eIF2α, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequestering it into a nonproductive complex.
The emergence of the SARS-CoV-2 Omicron variant is dominant in many countries worldwide. The high number of spike mutations is responsible for the broad immune evasion from existing vaccines and ...antibody drugs. To understand this, we first present the cryo-electron microscopy structure of ACE2-bound SARS-CoV-2 Omicron spike. Comparison to previous spike antibody structures explains how Omicron escapes these therapeutics. Secondly, we report structures of Omicron, Delta, and wild-type spikes bound to a patient-derived Fab antibody fragment (510A5), which provides direct evidence where antibody binding is greatly attenuated by the Omicron mutations, freeing spike to bind ACE2. Together with biochemical binding and 510A5 neutralization assays, our work establishes principles of binding required for neutralization and clearly illustrates how the mutations lead to antibody evasion yet retain strong ACE2 interactions. Structural information on spike with both bound and unbound antibodies collectively elucidates potential strategies for generation of therapeutic antibodies.
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•Structure of ACE2-bound Omicron spike explains its retained strong ACE2 interactions•Structure comparison reveals how Omicron escapes previous antibody therapeutics•The antibody-bound spike structures reveal the basis of 510A5 neutralizing mechanism•Omicron mutations greatly attenuate the antibody 510A5 binding and the neutralization
Guo et al. report the cryo-EM structures of ACE2-bound SARS-CoV-2 Omicron spike and human antibody (510A5)-bound spikes of Omicron, Delta, and WT. The structures clearly illustrate how Omicron mutations lead to antibody evasion yet retain strong ACE2 interactions.
The myxovirus resistance (Mx) proteins are interferon-induced dynamin GTPases that can inhibit a variety of viruses. Recently, MxB, but not MxA, was shown to restrict HIV-1 by an unknown mechanism ...that likely occurs in close proximity to the host cell nucleus and involves the viral capsid. Here, we present the crystal structure of MxB and reveal determinants involved in HIV-1 restriction. MxB adopts an extended antiparallel dimer and dimerization, but not higher-ordered oligomerization, is critical for restriction. Although MxB is structurally similar to MxA, the orientation of individual domains differs between MxA and MxB, and their antiviral functions rely on separate determinants, indicating distinct mechanisms for virus inhibition. Additionally, MxB directly binds the HIV-1 capsid, and this interaction depends on dimerization and the N terminus of MxB as well as the assembled capsid lattice. These insights establish a framework for understanding the mechanism by which MxB restricts HIV-1.
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•MxB forms an antiparallel dimer that is important for HIV-1 restriction•MxB interacts directly with HIV-1 capsid assemblies•MxB dimerization is important for restriction and its interaction with HIV-1 capsid•Higher-order MxB oligomerization is not essential for restriction or capsid binding
The MxB restriction factor has been shown to inhibit HIV-1 through an unknown mechanism. Fribourgh et al. present the crystal structure of the restriction factor MxB and demonstrate a direct interaction between MxB and HIV-1 capsid that requires the assembled capsid lattice. This study helps delineate the mechanism of HIV-1 restriction by MxB.
Dilated cardiomyopathy (DCM) is a common form of cardiomyopathy causing systolic dysfunction and heart failure. Rare variants in more than 30 genes, mostly encoding sarcomeric proteins and proteins ...of the cytoskeleton, have been implicated in familial DCM to date. Yet, the majority of variants causing DCM remain to be identified. The goal of the study is to identify novel mutations causing familial dilated cardiomyopathy.
We identify FBXO32 (ATROGIN 1), a member of the F-Box protein family, as a novel DCM-causing locus. The missense mutation affects a highly conserved amino acid and is predicted to severely impair binding to SCF proteins. This is validated by co-immunoprecipitation experiments from cells expressing the mutant protein and from human heart tissue from two of the affected patients. We also demonstrate that the hearts of the patients with the FBXO32 mutation show accumulation of selected proteins regulating autophagy.
Our results indicate that abnormal SCF activity with subsequent impairment of the autophagic flux due to a novel FBXO32 mutation is implicated in the pathogenesis of DCM.
Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified from mammalian proteins that bind lipid/sterol ligands via a hydrophobic pocket. In plants, ...predicted START domains are predominantly found in homeodomain leucine zipper (HD-Zip) transcription factors that are master regulators of cell-type differentiation in development. Here we utilized studies of Arabidopsis in parallel with heterologous expression of START domains in yeast to investigate the hypothesis that START domains are versatile ligand-binding motifs that can modulate transcription factor activity.
Our results show that deletion of the START domain from Arabidopsis Glabra2 (GL2), a representative HD-Zip transcription factor involved in differentiation of the epidermis, results in a complete loss-of-function phenotype, although the protein is correctly localized to the nucleus. Despite low sequence similarly, the mammalian START domain from StAR can functionally replace the HD-Zip-derived START domain. Embedding the START domain within a synthetic transcription factor in yeast, we found that several mammalian START domains from StAR, MLN64 and PCTP stimulated transcription factor activity, as did START domains from two Arabidopsis HD-Zip transcription factors. Mutation of ligand-binding residues within StAR START reduced this activity, consistent with the yeast assay monitoring ligand-binding. The D182L missense mutation in StAR START was shown to affect GL2 transcription factor activity in maintenance of the leaf trichome cell fate. Analysis of in vivo protein-metabolite interactions by mass spectrometry provided direct evidence for analogous lipid-binding activity in mammalian and plant START domains in the yeast system. Structural modeling predicted similar sized ligand-binding cavities of a subset of plant START domains in comparison to mammalian counterparts.
The START domain is required for transcription factor activity in HD-Zip proteins from plants, although it is not strictly necessary for the protein's nuclear localization. START domains from both mammals and plants are modular in that they can bind lipid ligands to regulate transcription factor function in a yeast system. The data provide evidence for an evolutionarily conserved mechanism by which lipid metabolites can orchestrate transcription. We propose a model in which the START domain is used by both plants and mammals to regulate transcription factor activity.
The Light-Dependent Protochlorophyllide Oxidoreductase (LPOR) catalyzes a crucial step in chlorophyll biosynthesis: the rare biological photocatalytic reduction of the double C&z.dbd;C bond in the ...precursor, protochlorophyllide (Pchlide). Despite its fundamental significance, limited structural insights into the active complex have hindered understanding of its reaction mechanism. Recently, a high-resolution cryo-EM structure of LPOR in its active conformation challenged our view of pigment binding, residue interactions, and the catalytic process. Surprisingly, this structure contrasts markedly with previous assumptions, particularly regarding the orientation of the bound Pchlide. To gain insights into the substrate binding puzzle, we conducted molecular dynamics simulations, quantum-mechanics/molecular-mechanics (QM/MM) calculations, and site-directed mutagenesis. Two Pchlide binding modes were considered, one aligning with historical proposals (mode A) and another consistent with the recent experimental data (mode B). Binding energy calculations revealed that in contrast to the non-specific interactions found for mode A, mode B exhibits distinct stabilizing interactions that support more thermodynamically favorable binding. A comprehensive analysis incorporating QM/MM-based local energy decomposition unraveled a complex interaction network involving Y177, H319, and the C13
1
carboxy group, influencing the pigment's excited state energy and potentially contributing to substrate specificity. Importantly, our results uniformly favor mode B, challenging established interpretations and emphasizing the need for a comprehensive re-evaluation of the LPOR reaction mechanism in a way that incorporates accurate structural information on pigment interactions and substrate-cofactor positioning in the binding pocket. The results shed light on the intricacies of LPOR's catalytic mechanism and provide a solid foundation for further elucidating the secrets of chlorophyll biosynthesis.
Two protochlorophyllide binding modes to the light-dependent protochlorophyllide oxidoreductase were considered: mode B exhibits more thermodynamically favorable binding.
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•The crystal structure of hnRNP A2/B1 RRMs dimer in complex with U-shaped conformation of ssDNA has been determined.•hnRNP A2/B1 RRMs homodimerise in solution by binding to ...pre-generated U-shaped DNA, either in ssDNA or dsDNA.•hnRNP A2/B1 RRMs homodimer induced by U-shaped ssDNA binding in solution exists a “Dimer A2” protein dimer interface.•The dimer interface formed in solution biochemically which depicts a potential functional state of hnRNP A2/B1.
Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been identified as a nuclear DNA sensor. Upon viral infection, hnRNP A2/B1 recognizes pathogen-derived DNA as a homodimer, which is a prerequisite for its translocation to the cytoplasm to activate the interferon response. However, the DNA binding mechanism inducing hnRNP A2/B1 homodimerization is unknown. Here, we show the crystal structure of the RNA recognition motif (RRM) of hnRNP A2/B1 in complex with a U-shaped ssDNA, which mediates the formation of a newly observed protein dimer. Our biochemical assays and mutagenesis studies confirm that the hnRNP A2/B1 homodimer forms in solution by binding to pre-generated ssDNA or dsDNA with a U-shaped bulge. These results depict a potential functional state of hnRNP A2/B1 in antiviral immunity and other cellular processes.
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