Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion.

Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.

Neutralization, effector function and immune imprinting of Omicron variants.

Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain1 (RBD) of the spike protein. The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting.

ATP-triggered conformational changes delineate substrate-binding and -folding mechanics of the GroEL chaperonin.

The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the "power stroke" that ejects substrate into the folding chamber.

Structural basis for isoform-specific inhibition of human CTPS1.

Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al], [Nature] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al], [Nature] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [J. Biol. Chem. ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al], [Nature] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al], [Nat. Struct. Mol. Biol.] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.

Light-sensitive phosphorylation regulates retinal IMPDH1 activity and filament assembly.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.

Broadly neutralizing antibodies against emerging delta-coronaviruses.

Porcine deltacoronavirus (PDCoV) spillovers were recently detected in children with acute undifferentiated febrile illness, underscoring recurrent zoonoses of divergent coronaviruses. To date, no vaccines or specific therapeutics are approved for use in humans against PDCoV. To prepare for possible future PDCoV epidemics, we isolated human spike (S)-directed monoclonal antibodies from transgenic mice and found that two of them, designated PD33 and PD41, broadly neutralized a panel of PDCoV variants. Cryo-electron microscopy structures of PD33 and PD41 in complex with the PDCoV receptor-binding domain and S ectodomain trimer provide a blueprint of the epitopes recognized by these mAbs, rationalizing their broad inhibitory activity. We show that both mAbs inhibit PDCoV by competitively interfering with host APN binding to the PDCoV receptor-binding loops, explaining the mechanism of viral neutralization. PD33 and PD41 are candidates for clinical advancement, which could be stockpiled to prepare for possible future PDCoV outbreaks.

Human PRPS1 filaments stabilize allosteric sites to regulate activity.

The universally conserved enzyme phosphoribosyl pyrophosphate synthetase (PRPS) assembles filaments in evolutionarily diverse organisms. PRPS is a key regulator of nucleotide metabolism, and mutations in the human enzyme PRPS1 lead to a spectrum of diseases. Here we determine structures of human PRPS1 filaments in active and inhibited states, with fixed assembly contacts accommodating both conformations. The conserved assembly interface stabilizes the binding site for the essential activator phosphate, increasing activity in the filament. Some disease mutations alter assembly, supporting the link between filament stability and activity. Structures of active PRPS1 filaments turning over substrate also reveal coupling of catalysis in one active site with product release in an adjacent site. PRPS1 filaments therefore provide an additional layer of allosteric control, conserved throughout evolution, with likely impact on metabolic homeostasis. Stabilization of allosteric binding sites by polymerization adds to the growing diversity of assembly-based enzyme regulatory mechanisms.

Discrimination of foreign bodies in quinoa (Chenopodium quinoa Willd.) grains using convolutional neural networks with a transfer learning approach.

The rising interest in quinoa (Chenopodium quinoa Willd.) is due to its high protein content and gluten-free condition; nonetheless, the presence of foreign bodies in quinoa processing facilities is an issue that must be addressed. As a result, convolutional neural networks have been adopted, mostly because of their data extraction capabilities, which had not been utilized before for this purpose. Consequently, the main objective of this work is to evaluate convolutional neural networks with a learning transfer for foreign bodies identification in quinoa samples. For experimentation, quinoa samples were collected and manually split into 17 classes: quinoa grains and 16 foreign bodies. Then, one thousand images were obtained from each class in RGB space and transformed into four different color spaces (L*a*b*, HSV, YCbCr, and Gray). Three convolutional neural networks (AlexNet, MobileNetv2, and DenseNet-201) were trained using the five color spaces, and the evaluation results were expressed in terms of accuracy and F-score. All the CNN approaches compared showed an F-score ranging from 98% to 99%; both color space and CNN structure were found to have significant effects on the F-score. Also, DenseNet-201 was the most robust architecture and, at the same time, the most time-consuming. These results evidence the capacity of CNN architectures to be used for the discrimination of foreign bodies in quinoa processing facilities.

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands.

Broad receptor tropism and immunogenicity of a clade 3 sarbecovirus.

Although Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S) glycoproteins remain uncharacterized. Here, we show that the African Rhinolophus bat clade 3 sarbecovirus PRD-0038 S has a broad angiotensin-converting enzyme 2 (ACE2) usage and that receptor-binding domain (RBD) mutations further expand receptor promiscuity and enable human ACE2 utilization. We determine a cryo-EM structure of the PRD-0038 RBD bound to Rhinolophus alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038 S using cryo-EM and monoclonal antibody reactivity reveals its distinct antigenicity relative to SARS-CoV-2 and identifies PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038 S vaccination elicits greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared with SARS-CoV-2 S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.

Broad receptor tropism and immunogenicity of a clade 3 sarbecovirus.

Although Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S) glycoproteins remain uncharacterized. Here, we show that the African Rinolophus bat clade 3 sarbecovirus PRD-0038 S has a broad ACE2 usage and that RBD mutations further expand receptor promiscuity and enable human ACE2 utilization. We determined a cryoEM structure of the PRD-0038 RBD bound to R. alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038 S using cryoEM and monoclonal antibody reactivity revealed its distinct antigenicity relative to SARS-CoV-2 and identified PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038 S vaccination elicited greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared to SARS-CoV-2 S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex.

We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alphaCTD), interactions of alphaCTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.

IMPDH1 retinal variants control filament architecture to tune allosteric regulation.

Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.

Block Liposome and Nanotube Formation is a General Phenomenon of Two-Component Membranes Containing Multivalent Lipids.

We report a study on the formation of block liposomes (BLs) and nanotubes from membranes comprised of mixtures of membrane curvature-stabilizing multivalent cationic lipids MVL3(3+) and MVL5(5+) with neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). In conjunction with prior work on MVLBG2(16+), our experiments suggest that BL and nanotube formation is a general phenomenon in membranes containing multivalent lipids, thus enhancing the relevance of BLs for applications such as gene/drug storage and delivery or templating.

Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation.

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

Coordination of phage genome degradation versus host genome protection by a bifunctional restriction-modification enzyme visualized by CryoEM.

Restriction enzymes that combine methylation and cleavage into a single assemblage and modify one DNA strand are capable of efficient adaptation toward novel targets. However, they must reliably cleave invasive DNA and methylate newly replicated unmodified host sites. One possible solution is to enforce a competition between slow methylation at a single unmodified host target, versus faster cleavage that requires multiple unmodified target sites in foreign DNA to be brought together in a reaction synapse. To examine this model, we have determined the catalytic behavior of a bifunctional type IIL restriction-modification enzyme and determined its structure, via cryoelectron microscopy, at several different stages of assembly and coordination with bound DNA targets. The structures demonstrate a mechanism in which an initial dimer is formed between two DNA-bound enzyme molecules, positioning the endonuclease domain from each enzyme against the other's DNA and requiring further additional DNA-bound enzyme molecules to enable cleavage.

Structural basis for broad coronavirus neutralization.

Three highly pathogenic ß-coronaviruses crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. SARS-CoV-2 has infected more than 64 million people worldwide, claimed over 1.4 million lives and is responsible for the ongoing COVID-19 pandemic. We isolated a monoclonal antibody, termed B6, cross-reacting with eight ß-coronavirus spike glycoproteins, including all five human-infecting ß-coronaviruses, and broadly inhibiting entry of pseudotyped viruses from two coronavirus lineages. Cryo-electron microscopy and X-ray crystallography characterization reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery and indicate that antibody binding sterically interferes with spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with ß-coronaviruses from three lineages along with proof-of-concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-coronavirus vaccine.

Structural basis for broad coronavirus neutralization.

Three highly pathogenic ß-coronaviruses have crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. To evaluate the possibility of identifying antibodies with broad neutralizing activity, we isolated a monoclonal antibody, termed B6, that cross-reacts with eight ß-coronavirus spike glycoproteins, including all five human-infecting ß-coronaviruses. B6 broadly neutralizes entry of pseudotyped viruses from lineages A and C, but not from lineage B, and the latter includes SARS-CoV and SARS-CoV-2. Cryo-EM, X-ray crystallography and membrane fusion assays reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery. The data indicate that antibody binding sterically interferes with the spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with ß-coronaviruses from three lineages, along with a proof of concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-ß-coronavirus vaccine.

A toolbox for ab initio 3-D reconstructions in single-particle electron microscopy.

Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a "toolbox" of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.