NINJ1 mediates plasma membrane rupture by cutting and releasing membrane disks.

The membrane protein NINJ1 mediates plasma membrane rupture in pyroptosis and other lytic cell death pathways. Here, we report the cryo-EM structure of a NINJ1 oligomer segmented from NINJ1 rings. Each NINJ1 subunit comprises amphipathic (⍺1, ⍺2) and transmembrane (TM) helices (⍺3, ⍺4) and forms a chain of subunits, mainly by the TM helices and ⍺1. ⍺3 and ⍺4 are kinked, and the Gly residues are important for function. The NINJ1 oligomer possesses a concave hydrophobic side that should face the membrane and a convex hydrophilic side formed by ⍺1 and ⍺2, presumably upon activation. This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. Live-cell and super-resolution imaging uncover ring-like structures on the plasma membrane that are released into the culture supernatant. Released NINJ1 encircles a membrane inside, as shown by lipid staining. Therefore, NINJ1-mediated membrane disk formation is different from gasdermin-mediated pore formation, resulting in membrane loss and plasma membrane rupture.

POT1 recruits and regulates CST-Polα/primase at human telomeres.

Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

FilamentID reveals the composition and function of metabolic enzyme polymers during gametogenesis.

Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.

Minimal and hybrid hydrogenases are active from archaea.

Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.

RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

Capturing totipotency in human cells through spliceosomal repression.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

Thiolase: A Versatile Biocatalyst Employing Coenzyme A-Thioester Chemistry for Making and Breaking C-C Bonds.

Thiolases are CoA-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA, as well as its reverse reaction, which is the thioester-dependent Claisen condensation reaction. Thiolases are dimers or tetramers (dimers of dimers). All thiolases have two reactive cysteines: (a) a nucleophilic cysteine, which forms a covalent intermediate, and (b) an acid/base cysteine. The best characterized thiolase is the Zoogloea ramigera thiolase, which is a bacterial biosynthetic thiolase belonging to the CT-thiolase subfamily. The thiolase active site is also characterized by two oxyanion holes, two active site waters, and four catalytic loops with characteristic amino acid sequence fingerprints. Three thiolase subfamilies can be identified, each characterized by a unique sequence fingerprint for one of their catalytic loops, which causes unique active site properties. Recent insights concerning the thiolase reaction mechanism, as obtained from recent structural studies, as well as from classical and recent enzymological studies, are addressed, and open questions are discussed.

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine.

ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.

Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice.

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

Receptor binding and complex structures of human ACE2 to spike RBD from omicron and delta SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues worldwide with many variants arising, some of which are variants of concern (VOCs). A recent VOC, omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here, we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryoelectron microscopy structures of the omicron RBD-hACE2 complex as well as the crystal structure of the delta RBD-hACE2 complex. We found that, unlike alpha, beta, and gamma, omicron RBD binds to hACE2 at a similar affinity to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of omicron RBD-hACE2 and delta RBD-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.

Structural analysis of the full-length human LRRK2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2G2019S. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.

Virus-encoded histone doublets are essential and form nucleosome-like structures.

The organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of "minimalist" histones in archaea and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones are essential for viral infectivity, localize to cytoplasmic viral factories after virus infection, and ultimately are found in the mature virions. Cryogenic electron microscopy (cryo-EM) structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of "organisms" that require nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses.

Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening.

Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.

Cyclic CMP and cyclic UMP mediate bacterial immunity against phages.

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.

Pan-ebolavirus protective therapy by two multifunctional human antibodies.

Ebolaviruses cause a severe and often fatal illness with the potential for global spread. Monoclonal antibody-based treatments that have become available recently have a narrow therapeutic spectrum and are ineffective against ebolaviruses other than Ebola virus (EBOV), including medically important Bundibugyo (BDBV) and Sudan (SUDV) viruses. Here, we report the development of a therapeutic cocktail comprising two broadly neutralizing human antibodies, rEBOV-515 and rEBOV-442, that recognize non-overlapping sites on the ebolavirus glycoprotein (GP). Antibodies in the cocktail exhibited synergistic neutralizing activity, resisted viral escape, and possessed differing requirements for their Fc-regions for optimal in vivo activities. The cocktail protected non-human primates from ebolavirus disease caused by EBOV, BDBV, or SUDV with high therapeutic effectiveness. High-resolution structures of the cocktail antibodies in complex with GP revealed the molecular determinants for neutralization breadth and potency. This study provides advanced preclinical data to support clinical development of this cocktail for pan-ebolavirus therapy.

The molecular basis for sarcomere organization in vertebrate skeletal muscle.

Sarcomeres are force-generating and load-bearing devices of muscles. A precise molecular picture of how sarcomeres are built underpins understanding their role in health and disease. Here, we determine the molecular architecture of native vertebrate skeletal sarcomeres by electron cryo-tomography. Our reconstruction reveals molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band, and Z-disc and demonstrates that α-actinin cross-links antiparallel actin filaments by forming doublets with 6-nm spacing. Structures of myosin, tropomyosin, and actin at ~10 Å further reveal two conformations of the "double-head" myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases.

The antigenic anatomy of SARS-CoV-2 receptor binding domain.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.

Bacterial Vipp1 and PspA are members of the ancient ESCRT-III membrane-remodeling superfamily.

Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.

Scap structures highlight key role for rotation of intertwined luminal loops in cholesterol sensing.

The cholesterol-sensing protein Scap induces cholesterol synthesis by transporting membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi apparatus for proteolytic activation. Transport requires interaction between Scap's two ER luminal loops (L1 and L7), which flank an intramembrane sterol-sensing domain (SSD). Cholesterol inhibits Scap transport by binding to L1, which triggers Scap's binding to Insig, an ER retention protein. Here we used cryoelectron microscopy (cryo-EM) to elucidate two structures of full-length chicken Scap: (1) a wild-type free of Insigs and (2) mutant Scap bound to chicken Insig without cholesterol. Strikingly, L1 and L7 intertwine tightly to form a globular domain that acts as a luminal platform connecting the SSD to the rest of Scap. In the presence of Insig, this platform undergoes a large rotation accompanied by rearrangement of Scap's transmembrane helices. We postulate that this conformational change halts Scap transport of SREBPs and inhibits cholesterol synthesis.