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Dynamin assembles as a helical polymer at the neck of budding endocytic vesicles, constricting the underlying membrane as it progresses through the GTPase cycle to sever vesicles from the plasma membrane. Although atomic models of the dynamin helical polymer bound to guanosine triphosphate (GTP) analogs define earlier stages of membrane constriction, there are no atomic models of the assembled state post-GTP hydrolysis. Here, we used cryo-EM methods to determine atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in a guanosine diphosphate (GDP)-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix with an inner lumen of 3.4 nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using the GTPase-defective dynamin K44A mutant and observed diverse dynamin helices, demonstrating that dynamin can accommodate a range of assembled complexes in cells that likely precede membrane fission., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Dominant optic atrophy is one of the leading causes of childhood blindness. Around 60-80% of cases 1 are caused by mutations of the gene that encodes optic atrophy protein 1 (OPA1), a protein that has a key role in inner mitochondrial membrane fusion and remodelling of cristae and is crucial for the dynamic organization and regulation of mitochondria 2 . Mutations in OPA1 result in the dysregulation of the GTPase-mediated fusion process of the mitochondrial inner and outer membranes 3 . Here we used cryo-electron microscopy methods to solve helical structures of OPA1 assembled on lipid membrane tubes, in the presence and absence of nucleotide. These helical assemblies organize into densely packed protein rungs with minimal inter-rung connectivity, and exhibit nucleotide-dependent dimerization of the GTPase domains-a hallmark of the dynamin superfamily of proteins 4 . OPA1 also contains several unique secondary structures in the paddle domain that strengthen its membrane association, including membrane-inserting helices. The structural features identified in this study shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly and membrane interactions. Furthermore, mutations that disrupt the assembly interfaces and membrane binding of OPA1 cause mitochondrial fragmentation in cell-based assays, providing evidence of the biological relevance of these interactions., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Human mitochondrial Hsp60 (mtHsp60) is a class I chaperonin, 51% identical in sequence to the prototypical E. coli chaperonin GroEL. mtHsp60 maintains the proteome within the mitochondrion and is associated with various neurodegenerative diseases and cancers. The oligomeric assembly of mtHsp60 into heptameric ring structures that enclose a folding chamber only occurs upon addition of ATP and is significantly more labile than that of GroEL, where the only oligomeric species is a tetradecamer. The lability of the mtHsp60 heptamer provides an opportunity to detect and visualize lower-order oligomeric states that may represent intermediates along the assembly/disassembly pathway. Using cryo-electron microscopy we show that, in addition to the fully-formed heptamer and an "inverted" tetradecamer in which the two heptamers associate via their apical domains, thereby blocking protein substrate access, well-defined lower-order oligomeric species, populated at less than 6% of the total particles, are observed. Specifically, we observe open trimers, tetramers, pentamers and hexamers (comprising ∼4% of the total particles) with rigid body rotations from one subunit to the next within ∼1.5-3.5° of that for the heptamer, indicating that these may lie directly on the assembly/disassembly pathway. We also observe a closed-ring hexamer (∼2% of the particles) which may represent an off-pathway species in the assembly/disassembly process in so far that conversion to the mature heptamer would require the closed-ring hexamer to open to accept an additional subunit. Lastly, we observe several classes of tetramers where additional subunits characterized by fuzzy electron density are caught in the act of oligomer extension., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

In Figs. 2b and 3d of this Letter, the labels 'Dynamin 1' and 'Overlay' were inadvertently swapped. This has been corrected online.

Membrane fission is a fundamental process in the regulation and remodelling of cell membranes. Dynamin, a large GTPase, mediates membrane fission by assembling around, constricting and cleaving the necks of budding vesicles 1 . Here we report a 3.75 Å resolution cryo-electron microscopy structure of the membrane-associated helical polymer of human dynamin-1 in the GMPPCP-bound state. The structure defines the helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays. Compared to the lipid-free tetramer form 2 , membrane-associated dynamin binds to the lipid bilayer with its pleckstrin homology domain (PHD) and self-assembles across the helical rungs via its guanine nucleotide-binding (GTPase) domain 3 . Notably, interaction with the membrane and helical assembly are accommodated by a severely bent bundle signalling element (BSE), which connects the GTPase domain to the rest of the protein. The BSE conformation is asymmetric across the inter-rung GTPase interface, and is unique compared to all known nucleotide-bound states of dynamin. The structure suggests that the BSE bends as a result of forces generated from the GTPase dimer interaction that are transferred across the stalk to the PHD and lipid membrane. Mutations that disrupted the BSE kink impaired endocytosis. We also report a 10.1 Å resolution cryo-electron microscopy map of a super-constricted dynamin polymer showing localized conformational changes at the BSE and GTPase domains, induced by GTP hydrolysis, that drive membrane constriction. Together, our results provide a structural basis for the mechanism of action of dynamin on the lipid membrane.

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs essential for animal germ cell development. Despite intense investigation of post-transcriptional processing, chromatin regulators for piRNA biogenesis in mammals remain largely unexplored. Here we document that BTBD18 is a pachytene nuclear protein in mouse testes that occupies a subset of pachytene piRNA-producing loci. Ablation of Btbd18 in mice disrupts piRNA biogenesis, prevents spermiogenesis, and results in male sterility. Transcriptome profiling, chromatin accessibility, and RNA polymerase II occupancy demonstrate that BTBD18 facilitates expression of pachytene piRNA precursors by promoting transcription elongation. Thus, our study identifies BTBD18 as a specific controller for transcription activation through RNA polymerase II elongation at a subset of genomic piRNA loci., (Published by Elsevier Inc.)

During development from oocyte to embryo, genetic programs in mouse germ cells are reshaped by chromatin remodeling to orchestrate the onset of development. Epigenetic modifications of specific amino acid residues of core histones and their isoforms can dramatically alter activation and suppression of gene expression. H3.3 is a histone H3 variant that plays essential roles in mouse oocytes and early embryos, but the functional role of individual amino acid residues has been unclear because of technical hurdles. Here, we describe two strategies that successfully investigated the functions of three individual H3.3 residues in oogenesis, cleavage-stage embryogenesis and early development. We first generated genetic mosaic ovaries and blastocysts with stochastic expression of wild-type or mutant H3.3 alleles and showed dominant negative effects of H3.3R26 and H3.3K27 in modulating oogenesis and partitioning cells to the inner cell mass of the early embryo. Time-lapse imaging assays also revealed the essential roles of H3.3K56 in efficient H2B incorporation and paternal pronuclei formation. Application of these strategies can be extended to investigate roles of additional H3.3 residues and has implications for use in other developmental systems., (© 2017. Published by The Company of Biologists Ltd.)

The ESCRT-I and ESCRT-II supercomplex induces membrane buds that invaginate into the lumen of endosomes, a process central to the lysosomal degradation of ubiquitinated membrane proteins. The solution conformation of the membrane-budding ESCRT-I-II supercomplex from yeast was refined against small-angle X-ray scattering (SAXS), single-molecule Förster resonance energy transfer (smFRET), and double electron-electron resonance (DEER) spectra. These refinements yielded an ensemble of 18 ESCRT-I-II supercomplex structures that range from compact to highly extended. The crescent shapes of the ESCRT-I-II supercomplex structures provide the basis for a detailed mechanistic model, in which ESCRT-I-II stabilizes membrane buds and coordinates cargo sorting by lining the pore of the nascent bud necks. The hybrid refinement used here is general and should be applicable to other dynamic multiprotein assmeblies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Although receptor-mediated regulation of small G-proteins and the cytoskeleton is intensively studied, the mechanisms for attenuation of these signals are poorly understood. In this study, we have identified the Rac-GAP beta2-chimaerin as an effector of the epidermal growth factor receptor (EGFR) via coupling to phospholipase Cgamma (PLCgamma) and generation of the lipid second messenger diacylglycerol (DAG). EGF redistributes beta2-chimaerin to promote its association with the small GTPase Rac1 at the plasma membrane, as determined by FRET. This relocalization and association with Rac1 were impaired by disruption of the beta2-chimaerin C1 domain as well as by PLCgamma1 RNAi, thus defining beta2-chimaerin as a novel DAG effector. On the other hand, GAP-deficient beta2-chimaerin mutants show enhanced translocation and sustained Rac1 association in the FRET assays. Remarkably, RNAi depletion of beta2-chimaerin significantly extended the duration of Rac activation by EGF, suggesting that beta2-chimaerin serves as a mechanism that self-limits Rac activity in response to EGFR activation. Our results represent the first direct evidence of divergence in DAG signaling downstream of a tyrosine-kinase receptor via a PKC-independent mechanism.

The lipid second messenger diacylglycerol acts by binding to the C1 domains of target proteins, which translocate to cell membranes and are allosterically activated. Here we report the crystal structure at 3.2 A resolution of one such protein, beta2-chimaerin, a GTPase-activating protein for the small GTPase Rac, in its inactive conformation. The structure shows that in the inactive state, the N terminus of beta2-chimaerin protrudes into the active site of the RacGAP domain, sterically blocking Rac binding. The diacylglycerol and phospholipid membrane binding site on the C1 domain is buried by contacts with the four different regions of beta2-chimaerin: the N terminus, SH2 domain, RacGAP domain, and the linker between the SH2 and C1 domains. Phospholipid binding to the C1 domain triggers the cooperative dissociation of these interactions, allowing the N terminus to move out of the active site and thereby activating the enzyme.

Many novel signal transduction domains are being identified in the wake of genome sequencing projects and improved sensitivity in homology-detection techniques. The functions of these domains are being discovered by hypothesis-driven experiments and structural genomics approaches. This article reviews the recent highlights of research on modular signaling domains, and the relative contributions and limitations of the various approaches being used.

Background: The reactive center loops (RCL) of serpins undergo large conformational changes triggered by the interaction with their target protease. Available crystallographic data suggest that the serpin RCL is polymorphic, but the relevance of the observed conformations to the competent active structure and the conformational changes that occur on binding target protease has remained obscure. New high-resolution data on an active serpin, serpin 1K from the moth hornworm Manduca sexta, provide insights into how active serpins are stabilized and how conformational changes are induced by protease binding., Results: The 2.1 A structure shows that the RCL of serpin 1K, like that of active alpha1-antitrypsin, is canonical, complimentary and ready to bind to the target protease between P3 and P3 (where P refers to standard protease nomenclature),. In the hinge region (P17-P13), however, the RCL of serpin 1K, like ovalbumin and alpha1-antichymotrypsin, forms tight interactions that stabilize the five-stranded closed form of betasheet A. These interactions are not present in, and are not compatible with, the observed structure of active alpha1-antitrypsin., Conclusions: Serpin 1K may represent the best resting conformation for serpins - canonical near P1, but stabilized in the closed conformation of betasheet A. By comparison with other active serpins, especially alpha1-antitrypsin, a model is proposed in which interaction with the target protease near P1 leads to conformational changes in betasheet A of the serpin.

Background: The mitogen-activated protein (MAP) kinases are important signaling molecules that participate in diverse cellular events and are potential targets for intervention in inflammation, cancer, and other diseases. The MAP kinase p38 is responsive to environmental stresses and is involved in the production of cytokines during inflammation. In contrast, the activation of the MAP kinase ERK2 (extracellular-signal-regulated kinase 2) leads to cellular differentiation or proliferation. The anti-inflammatory agent pyridinylimidazole and its analogs (SB [SmithKline Beecham] compounds) are highly potent and selective inhibitors of p38, but not of the closely-related ERK2, or other serine/threonine kinases. Although these compounds are known to bind to the ATP-binding site, the origin of the inhibitory specificity toward p38 is not clear., Results: We report the structural basis for the exceptional selectivity of these SB compounds for p38 over ERK2, as determined by comparative crystallography. In addition, structural data on the origin of olomoucine (a better inhibitor of ERK2) selectivity are presented. The crystal structures of four SB compounds in complex with p38 and of one SB compound and olomoucine in complex with ERK2 are presented here. The SB inhibitors bind in an extended pocket in the active site and are complementary to the open domain structure of the low-activity form of p38. The relatively closed domain structure of ERK2 is able to accommodate the smaller olomoucine., Conclusions: The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.

The MAP kinase ERK2 is widely involved in eukaryotic signal transduction. Upon activation it translocates to the nucleus of the stimulated cell, where it phosphorylates nuclear targets. We find that nuclear accumulation of microinjected ERK2 depends on its phosphorylation state rather than on its activity or on upstream components of its signaling pathway. Phosphorylated ERK2 forms dimers with phosphorylated and unphosphorylated ERK2 partners. Disruption of dimerization by mutagenesis of ERK2 reduces its ability to accumulate in the nucleus, suggesting that dimerization is essential for its normal ligand-dependent relocalization. The crystal structure of phosphorylated ERK2 reveals the basis for dimerization. Other MAP kinase family members also form dimers. The generality of this behavior suggests that dimerization is part of the mechanism of action of the MAP kinase family.

The structure of the active form of the MAP kinase ERK2 has been solved, phosphorylated on a threonine and a tyrosine residue within the phosphorylation lip. The lip is refolded, bringing the phosphothreonine and phosphotyrosine into alignment with surface arginine-rich binding sites. Conformational changes occur in the lip and neighboring structures, including the P+1 site, the MAP kinase insertion, the C-terminal extension, and helix C. Domain rotation and remodeling of the proline-directed P+1 specificity pocket account for the activation. The conformation of the P+1 pocket is similar to a second proline-directed kinase, CDK2-CyclinA, thus permitting the origin of this specificity to be defined. Conformational changes outside the lip provide loci at which the state of phosphorylation can be felt by other cellular components.

The human basal transcription factor TFIIH plays a central role in two distinct processes. TFIIH is an obligatory component of the RNA polymerase II (RNAP II) transcription initiation complex. Additionally, it is believed to be the core structure around which some if not all the components of the nucleotide excision repair (NER) machinery assemble to constitute a nucleotide excision repairosome. At least two of the subunits of TFIIH (XPB and XPD proteins) are implicated in the disease xeroderma pigmentosum (XP). We have exploited the availability of the cloned XPB, XPD, p62, p44, and p34 genes (all of which encode polypeptide subunits of TFIIH) to examine interactions between in vitro-translated polypeptides by co-immunoprecipitation. Additionally we have examined interactions between TFIIH components, the human NER protein XPG, and the CSB protein which is implicated in Cockayne syndrome (CS). Our analyses demonstrate that the XPB, XPD, p44, and p62 proteins interact with each other. XPG protein interacts with multiple subunits of TFIIH and with CSB protein.

Trypanosoma brucei ornithine decarboxylase was reconstituted by coexpression of two polypeptides corresponding to residues 1-305 and residues 306-425 in Escherichia coli. The two peptides were coexpressed, at wild-type levels, from a single transcriptional unit that was separated by a 15-nucleotide untranslated region containing a ribosome binding site. The fragmented enzyme was purified and analyzed. The N- and C-terminal peptides are tightly associated into a fully active tetramer which has the same molecular weight as the native dimer. The kinetic constants (Km and kcat) measured for the decarboxylation of ornithine are identical to those obtained for the wild-type enzyme. These results suggest that the enzyme is organized into two structural domains, with a domain boundary in the region of amino acid 305. In contrast, the individual N- and C-terminal peptides are expressed primarily as inclusion bodies. Small quantities of soluble N-terminal peptide could be purified. This truncated protein is capable of inhibiting the wild-type enzyme, suggesting that it is folded into a native-like structure. Limited proteolysis with trypsin or chymotrypsin identifies a likely surface loop at amino acids 160-170, present in both the mouse and T. brucei enzyme, which positions one or more functionally important active site residues (e.g., Lys169). Kinetic analysis of a chimeric enzyme composed of T. brucei and mouse ornithine decarboxylase suggests that the substrate carboxylate binding determinant is located between residues 1 and 170.