Structural characterization of antibody-responses from Zolgensma treatment provides the blueprint for the engineering of an AAV capsid suitable for redosing.

Monoclonal antibodies (mAbs) are useful tools to dissect the neutralizing antibody response against the adeno-associated virus (AAV) capsids used as gene therapy delivery vectors. This study structurally characterizes the interactions of 21 human-derived antibodies from patients treated with the AAV9 vector, Zolgensma ® , utilizing high-resolution cryo-electron microscopy. The majority of the bound antibodies do not conform to the icosahedral symmetry of the capsid, thus requiring localized reconstructions. These complex structures provide unprecedented details of the mAbs binding interfaces, with some antibodies inducing structural perturbations of the capsid upon binding. Key surface capsid amino acid residues were identified facilitating the design of capsid variants with an antibody escape phenotype, with the potential to expand the patient cohort treatable with AAV9 vectors to include those that were previously excluded due to their pre-existing neutralizing antibodies, and possibly also to those requiring redosing.

Structure and interactions of the endogenous human Commander complex.

The Commander complex, a 16-protein assembly, plays multiple roles in cell homeostasis, cell cycle and immune response. It consists of copper-metabolism Murr1 domain proteins (COMMD1-10), coiled-coil domain-containing proteins (CCDC22 and CCDC93), DENND10 and the Retriever subcomplex (VPS26C, VPS29 and VPS35L), all expressed ubiquitously in the body and linked to various diseases. Here, we report the structure and key interactions of the endogenous human Commander complex by cryogenic-electron microscopy and mass spectrometry-based proteomics. The complex consists of a stable core of COMMD1-10 and an effector containing DENND10 and Retriever, scaffolded together by CCDC22 and CCDC93. We establish the composition of Commander and reveal major interaction interfaces. These findings clarify its roles in intracellular transport, and uncover a strong association with cilium assembly, and centrosome and centriole functions.

Nanobody engineering for SARS-CoV-2 neutralization and detection.

In response to the ongoing COVID-19 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, i.e., "nanobodies." Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants at high potency, with IC50 values as low as 50 pM. Despite simultaneous binding to distinct epitopes, Beta and Omicron variants were more resistant to neutralization by the multimodular nanobodies, which highlights the importance of accounting for antigenic drift in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present an assay based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious diseases. IMPORTANCE: Nanobodies, small protein binders derived from the camelid antibody, are highly potent inhibitors of respiratory viruses that offer several advantages over conventional antibodies as candidates for specific therapies, including high stability and low production costs. In this work, we leverage the unique properties of nanobodies and apply them as building blocks for new therapeutic and diagnostic tools. We report ultra-potent SARS-CoV-2 inhibition by engineered nanobodies comprising multiple modules in structure-guided combinations and develop nanobodies that carry signal molecules, allowing rapid detection of the SARS-CoV-2 spike protein. Our results highlight the potential of engineered nanobodies in the development of effective countermeasures, both therapeutic and diagnostic, to manage outbreaks of emerging viruses.

Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Community recommendations on cryoEM data archiving and validation: Outcomes of a wwPDB/EMDB workshop on cryoEM data management, deposition and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and consensus recommendations resulting from the workshop. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Molecular view of ER membrane remodeling by the Sec61/TRAP translocon.

Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61 protein-conducting channel facilitates polypeptide translocation and coordinates cotranslational polypeptide-processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon-associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation remains unknown. Here, we present the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by cryogenic electron microscopy (cryo-EM). Ribosome interactions anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its lumenal leaflet. We propose that TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen mediated by direct polypeptide interactions.

DNA-origami-directed virus capsid polymorphism.

Viral capsids can adopt various geometries, most iconically characterized by icosahedral or helical symmetries. Importantly, precise control over the size and shape of virus capsids would have advantages in the development of new vaccines and delivery systems. However, current tools to direct the assembly process in a programmable manner are exceedingly elusive. Here we introduce a modular approach by demonstrating DNA-origami-directed polymorphism of single-protein subunit capsids. We achieve control over the capsid shape, size and topology by employing user-defined DNA origami nanostructures as binding and assembly platforms, which are efficiently encapsulated within the capsid. Furthermore, the obtained viral capsid coatings can shield the encapsulated DNA origami from degradation. Our approach is, moreover, not limited to a single type of capsomers and can also be applied to RNA-DNA origami structures to pave way for next-generation cargo protection and targeting strategies.

Signal peptide mimicry primes Sec61 for client-selective inhibition.

Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.

Intranasal trimeric sherpabody inhibits SARS-CoV-2 including recent immunoevasive Omicron subvariants.

The emergence of increasingly immunoevasive SARS-CoV-2 variants emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Intranasal administration of neutralizing antibodies has shown encouraging protective potential but there remains a need for SARS-CoV-2 blocking agents that are less vulnerable to mutational viral variation and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and stable trimeric antibody-mimetic sherpabody targeted against a conserved region of the viral spike glycoprotein. TriSb92 potently neutralizes SARS-CoV-2, including the latest Omicron variants like BF.7, XBB, and BQ.1.1. In female Balb/c mice intranasal administration of just 5 or 50 micrograms of TriSb92 as early as 8 h before but also 4 h after SARS-CoV-2 challenge can protect from infection. Cryo-EM and biochemical studies reveal triggering of a conformational shift in the spike trimer as the inhibitory mechanism of TriSb92. The potency and robust biochemical properties of TriSb92 together with its resistance against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.

MANF regulates neuronal survival and UPR through its ER-located receptor IRE1α.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in neurons and pancreatic ß cells in vitro and in models of neurodegeneration and diabetes in vivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1α, and we identify the binding interface between MANF and IRE1α. The expression of wild-type MANF, but not its IRE1α binding-deficient mutant, attenuates UPR signaling by decreasing IRE1α oligomerization; phosphorylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1α interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons in vitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1α as an intracellular receptor for MANF and regulator of neuronal survival.

Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms.

The actin cytoskeleton is critical for cell migration, morphogenesis, endocytosis, organelle dynamics, and cytokinesis. To support diverse cellular processes, actin filaments form a variety of structures with specific architectures and dynamic properties. Key proteins specifying actin filaments are tropomyosins. Non-muscle cells express several functionally non-redundant tropomyosin isoforms, which differentially control the interactions of other proteins, including myosins and ADF/cofilin, with actin filaments. However, the underlying molecular mechanisms have remained elusive. By determining the cryogenic electron microscopy structures of actin filaments decorated by two functionally distinct non-muscle tropomyosin isoforms, Tpm1.6 and Tpm3.2, we reveal that actin filament conformation remains unaffected upon binding. However, Tpm1.6 and Tpm3.2 follow different paths along the actin filament major groove, providing an explanation for their incapability to co-polymerize on actin filaments. We also elucidate the molecular basis underlying specific roles of Tpm1.6 and Tpm3.2 in myosin II activation and protecting actin filaments from ADF/cofilin-catalyzed severing.

Cryo-EM structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution.

The origin of viruses remains an open question. While lack of detectable sequence similarity hampers the analysis of distantly related viruses, structural biology investigations of conserved capsid protein structures facilitate the study of distant evolutionary relationships. Here we characterize the lipid-containing ssDNA temperate bacteriophage ΦCjT23, which infects Flavobacterium sp. (Bacteroidetes). We report ΦCjT23-like sequences in the genome of strains belonging to several Flavobacterium species. The virion structure determined by cryogenic electron microscopy reveals similarities to members of the viral kingdom Bamfordvirae that currently consists solely of dsDNA viruses with a major capsid protein composed of two upright ß-sandwiches. The minimalistic structure of ΦCjT23 suggests that this phage serves as a model for the last common ancestor between ssDNA and dsDNA viruses in the Bamfordvirae. Both ΦCjT23 and the related phage FLiP infect Flavobacterium species found in several environments, suggesting that these types of viruses have a global distribution and a shared evolutionary origin. Detailed comparisons to related, more complex viruses not only expand our knowledge about this group of viruses but also provide a rare glimpse into early virus evolution.

Mechanistic Insights into the Activation of Lecithin-Cholesterol Acyltransferase in Therapeutic Nanodiscs Composed of Apolipoprotein A-I Mimetic Peptides and Phospholipids.

The mechanistic details behind the activation of lecithin-cholesterol acyltransferase (LCAT) by apolipoprotein A-I (apoA-I) and its mimetic peptides are still enigmatic. Resolving the fundamental principles behind LCAT activation will facilitate the design of advanced HDL-mimetic therapeutic nanodiscs for LCAT deficiencies and coronary heart disease and for several targeted drug delivery applications. Here, we have combined coarse-grained molecular dynamics simulations with complementary experiments to gain mechanistic insight into how apoA-Imimetic peptide 22A and its variants tune LCAT activity in peptide-lipid nanodiscs. Our results highlight that peptide 22A forms transient antiparallel dimers in the rim of nanodiscs. The dimerization tendency considerably decreases with the removal of C-terminal lysine K22, which has also been shown to reduce the cholesterol esterification activity of LCAT. In addition, our simulations revealed that LCAT prefers to localize to the rim of nanodiscs in a manner that shields the membrane-binding domain (MBD), αA-αA', and the lid amino acids from the water phase, following previous experimental evidence. Meanwhile, the location and conformation of LCAT in the rim of nanodiscs are spatially more restricted when the active site covering the lid of LCAT is in the open form. The average location and spatial dimensions of LCAT in its open form were highly compatible with the electron microscopy images. All peptide 22A variants studied here had a specific interaction site in the open LCAT structure flanked by the lid and MBD domain. The bound peptides showed different tendencies to form antiparallel dimers and, interestingly, the temporal binding site occupancies of the peptide variants affected their in vitro ability to promote LCAT-mediated cholesterol esterification.

Structural basis of rapid actin dynamics in the evolutionarily divergent Leishmania parasite.

Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of the 'ancient' actin turnover machineries is limited. We show that, despite > 1 billion years of evolution, pathogenic Leishmania major parasite and mammalian actins share the same overall fold and co-polymerize with each other. Interestingly, Leishmania harbors a simple actin-regulatory machinery that lacks cofilin 'cofactors', which accelerate filament disassembly in higher eukaryotes. By applying single-filament biochemistry we discovered that, compared to mammalian proteins, Leishmania actin filaments depolymerize more rapidly from both ends, and are severed > 100-fold more efficiently by cofilin. Our high-resolution cryo-EM structures of Leishmania ADP-, ADP-Pi- and cofilin-actin filaments identify specific features at actin subunit interfaces and cofilin-actin interactions that explain the unusually rapid dynamics of parasite actin filaments. Our findings reveal how divergent parasites achieve rapid actin dynamics using a remarkably simple set of actin-binding proteins, and elucidate evolution of the actin cytoskeleton.

Snapshots of actin and tubulin folding inside the TRiC chaperonin.

The integrity of a cell's proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.

Structure of a Cell Entry Defective Human Adenovirus Provides Insights into Precursor Proteins and Capsid Maturation.

Maturation of adenoviruses is distinguished by proteolytic processing of several interior minor capsid proteins and core proteins by the adenoviral protease and subsequent reorganization of adenovirus core. We report the results derived from the icosahedrally averaged cryo-EM structure of a cell entry defective form of adenovirus, designated ts1, at a resolution of 3.7 Å as well as of the localized reconstructions of unique hexons and penton base. The virion structure revealed the structures and organization of precursors of minor capsid proteins, pIIIa, pVI and pVIII, which are closely associated with the hexons on the capsid interior. In addition to a well-ordered helical domain (a.a. 310-397) of pIIIa, highlights of the structure include the precursors of VIII display significantly different structures near the cleavage sites. Moreover, we traced residues 4-96 of the membrane lytic protein (pVI) that includes an amphipathic helix occluded deep in the hexon cavity suggesting the possibility of co-assembly of hexons with the precursors of VI. In addition, we observe a second copy of pVI ordered up to residue L40 in the peripentonal hexons and a few fragments of density corresponding to 2nd and 3rd copies of pVI in other hexons. However, we see no evidence of precursors of VII binding in the hexon cavity. These findings suggest the possibility that differently bound pVI molecules undergo processing at the N-terminal cleavage sites at varying efficiencies, subsequently creating competition between the cleaved and uncleaved forms of VI, followed by reorganization, processing, and release of VI molecules from the hexon cavities.

Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen.

Hantaviruses are a group of emerging pathogens capable of causing severe disease upon zoonotic transmission to humans. The mature hantavirus surface presents higher-order tetrameric assemblies of two glycoproteins, Gn and Gc, which are responsible for negotiating host cell entry and constitute key therapeutic targets. Here, we demonstrate that recombinantly derived Gn from Hantaan virus (HTNV) elicits a neutralizing antibody response (serum dilution that inhibits 50% infection [ID50], 1:200 to 1:850) in an animal model. Using antigen-specific B cell sorting, we isolated monoclonal antibodies (mAbs) exhibiting neutralizing and non-neutralizing activity, termed mAb HTN-Gn1 and mAb nnHTN-Gn2, respectively. Crystallographic analysis reveals that these mAbs target spatially distinct epitopes at disparate sites of the N-terminal region of the HTNV Gn ectodomain. Epitope mapping onto a model of the higher order (Gn-Gc)4 spike supports the immune accessibility of the mAb HTN-Gn1 epitope, a hypothesis confirmed by electron cryo-tomography of the antibody with virus-like particles. These data define natively exposed regions of the hantaviral Gn that can be targeted in immunogen design. IMPORTANCE The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits. We isolated a neutralizing (HTN-Gn1) and a non-neutralizing (nnHTN-Gn2) monoclonal antibody and provide the first molecular-level insights into how the Gn glycoprotein may be targeted by the antibody-mediated immune response. These findings may guide rational vaccine design approaches focused on targeting the hantavirus glycoprotein envelope.

Localized reconstruction in Scipion expedites the analysis of symmetry mismatches in cryo-EM data.

Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex. However, data processing that applies symmetry can average out asymmetric features and consequently data analysis methods are required to resolve asymmetric structural features. Scipion is a cryo-EM image processing framework that integrates functions from different image processing packages as plugins. To extend its functionality for handling symmetry mismatches, we present here a Scipion plugin termed LocalRec implementing the localized reconstruction method. When tested on an adenovirus data set, the plugin enables resolving the symmetry-mismatched trimeric fibre bound to the five-fold vertices of the capsid. Furthermore, it improves the structure determination of the icosahedral capsid by dealing with the defocus gradient across the particle. LocalRec is expected to be widely applicable in a range of cryo-EM investigations of flexible and symmetry mismatched complexes.

Molecular rationale for antibody-mediated targeting of the hantavirus fusion glycoprotein.

The intricate lattice of Gn and Gc glycoprotein spike complexes on the hantavirus envelope facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Through study of a neutralizing monoclonal antibody termed mAb P-4G2, which neutralizes the zoonotic pathogen Puumala virus (PUUV), we provide a molecular-level basis for antibody-mediated targeting of the hantaviral glycoprotein lattice. Crystallographic analysis demonstrates that P-4G2 binds to a multi-domain site on PUUV Gc and may preclude fusogenic rearrangements of the glycoprotein that are required for host-cell entry. Furthermore, cryo-electron microscopy of PUUV-like particles in the presence of P-4G2 reveals a lattice-independent configuration of the Gc, demonstrating that P-4G2 perturbs the (Gn-Gc)4 lattice. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.