Distinct ultrastructural phenotypes of glial and neuronal alpha-synuclein inclusions in multiple system atrophy.

Multiple System Atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy.

Structure-function analysis of the cyclic ß-1,2-glucan synthase from Agrobacterium tumefaciens.

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic ß-1,2-glucan (CßG) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). CßG is produced by the cyclic glucan synthase (Cgs), a multi-domain membrane protein. So far, its structure as well as the mechanism underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We determine the structure of this complex protein machinery and clarify key aspects of CßG synthesis, revealing a distinct mechanism that uses a tyrosine-linked oligosaccharide intermediate in cycles of polymerization and processing of the glucan chain. Our research opens possibilities for combating pathogens that rely on polysaccharide virulence factors and may lead to synthetic biology approaches for producing complex cyclic sugars.

Computational drug development for membrane protein targets.

The application of computational biology in drug development for membrane protein targets has experienced a boost from recent developments in deep learning-driven structure prediction, increased speed and resolution of structure elucidation, machine learning structure-based design and the evaluation of big data. Recent protein structure predictions based on machine learning tools have delivered surprisingly reliable results for water-soluble and membrane proteins but have limitations for development of drugs that target membrane proteins. Structural transitions of membrane proteins have a central role during transmembrane signaling and are often influenced by therapeutic compounds. Resolving the structural and functional basis of dynamic transmembrane signaling networks, especially within the native membrane or cellular environment, remains a central challenge for drug development. Tackling this challenge will require an interplay between experimental and computational tools, such as super-resolution optical microscopy for quantification of the molecular interactions of cellular signaling networks and their modulation by potential drugs, cryo-electron microscopy for determination of the structural transitions of proteins in native cell membranes and entire cells, and computational tools for data analysis and prediction of the structure and function of cellular signaling networks, as well as generation of promising drug candidates.

Structural basis of bile salt extrusion and small-molecule inhibition in human BSEP.

BSEP (ABCB11) is an ATP-binding cassette transporter that is expressed in hepatocytes and extrudes bile salts into the canaliculi of the liver. BSEP dysfunction, caused by mutations or induced by drugs, is frequently associated with severe cholestatic liver disease. We report the cryo-EM structure of glibenclamide-bound human BSEP in nanodiscs, revealing the basis of small-molecule inhibition. Glibenclamide binds the apex of a central binding pocket between the transmembrane domains, preventing BSEP from undergoing conformational changes, and thus rationalizing the reduced uptake of bile salts. We further report two high-resolution structures of BSEP trapped in distinct nucleotide-bound states by using a catalytically inactivated BSEP variant (BSEPE1244Q) to visualize a pre-hydrolysis state, and wild-type BSEP trapped by vanadate to visualize a post-hydrolysis state. Our studies provide structural and functional insight into the mechanism of bile salt extrusion and into small-molecule inhibition of BSEP, which may rationalize drug-induced liver toxicity.

Structure of a membrane-bound menaquinol:organohalide oxidoreductase.

Organohalide-respiring bacteria are key organisms for the bioremediation of soils and aquifers contaminated with halogenated organic compounds. The major players in this process are respiratory reductive dehalogenases, corrinoid enzymes that use organohalides as substrates and contribute to energy conservation. Here, we present the structure of a menaquinol:organohalide oxidoreductase obtained by cryo-EM. The membrane-bound protein was isolated from Desulfitobacterium hafniense strain TCE1 as a PceA2B2 complex catalysing the dechlorination of tetrachloroethene. Two catalytic PceA subunits are anchored to the membrane by two small integral membrane PceB subunits. The structure reveals two menaquinone molecules bound at the interface of the two different subunits, which are the starting point of a chain of redox cofactors for electron transfer to the active site. In this work, the structure elucidates how energy is conserved during organohalide respiration in menaquinone-dependent organohalide-respiring bacteria.

Structural Basis of the Allosteric Inhibition of Human ABCG2 by Nanobodies.

ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.

Fully co-factor-free ClearTau platform produces seeding-competent Tau fibrils for reconstructing pathological Tau aggregates.

Tau protein fibrillization is implicated in the pathogenesis of several neurodegenerative diseases collectively known as Tauopathies. For decades, investigating Tau fibrillization in vitro has required the addition of polyanions or other co-factors to induce its misfolding and aggregation, with heparin being the most commonly used. However, heparin-induced Tau fibrils exhibit high morphological heterogeneity and a striking structural divergence from Tau fibrils isolated from Tauopathies patients' brains at ultra- and macro-structural levels. To address these limitations, we developed a quick, cheap, and effective method for producing completely co-factor-free fibrils from all full-length Tau isoforms and mixtures thereof. We show that Tau fibrils generated using this ClearTau method - ClearTau fibrils - exhibit amyloid-like features, possess seeding activity in biosensor cells and hiPSC-derived neurons, retain RNA-binding capacity, and have morphological properties and structures more reminiscent of the properties of the brain-derived Tau fibrils. We present the proof-of-concept implementation of the ClearTau platform for screening Tau aggregation-modifying compounds. We demonstrate that these advances open opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies.

Activation mechanism of a short argonaute-TIR prokaryotic immune system.

Short prokaryotic argonaute (pAgo) and toll/interleukin-1 receptor/resistance protein (TIR)-analog of PAZ (APAZ) form a heterodimeric SPARTA complex that provides immunity to its prokaryotic host through an abortive infection mechanism. Monomeric SPARTA senses foreign RNA/DNA duplexes to assemble an active tetramer resulting in cell death by nicotinamide adenine dinucleotide (oxidized form) (NAD) depletion via an unknown mechanism. We report nine structures of SPARTA in different functional states at a resolution range of 4.2 to 2.9 angstroms, revealing its activation mechanism. Inactive SPARTA monomers bind to RNA/DNA duplexes to form symmetric dimers mediated by the association of Ago subunits. The initiation of tetramer assembly induces flexibility of the TIR domains enabling a symmetry-breaking rotational movement of a TIR domain in the dimer units which facilitates the TIR oligomerization, resulting in the formation of the substrate binding pocket and the activation of the SPARTA complex's NADase activity. Our findings provide detailed structural and mechanistic insights into activating a short argonaute defense system.

De novo design of protein interactions with learned surface fingerprints.

Physical interactions between proteins are essential for most biological processes governing life1. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic and structural data increase. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications2-9. Here we use a geometric deep-learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features that are critical to drive protein-protein interactions10. We hypothesized that these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof of principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1 and CTLA-4. Several designs were experimentally optimized, whereas others were generated purely in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling an approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range.

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected. Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors. Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor.

Structural insights into the regulation of Cas7-11 by TPR-CHAT.

The CRISPR-guided caspase (Craspase) complex is an assembly of the target-specific RNA nuclease known as Cas7-11 bound to CRISPR RNA (crRNA) and an ancillary protein known as TPR-CHAT (tetratricopeptide repeats (TPR) fused with a CHAT domain). The Craspase complex holds promise as a tool for gene therapy and biomedical research, but its regulation is poorly understood. TPR-CHAT regulates Cas7-11 nuclease activity via an unknown mechanism. In the present study, we use cryoelectron microscopy to determine structures of the Desulfonema magnum (Dm) Craspase complex to gain mechanistic insights into its regulation. We show that DmTPR-CHAT stabilizes crRNA-bound DmCas7-11 in a closed conformation via a network of interactions mediated by the DmTPR-CHAT N-terminal domain, the DmCas7-11 insertion finger and Cas11-like domain, resulting in reduced target RNA accessibility and cleavage.

Mechanism of cyclic ß-glucan export by ABC transporter Cgt of Brucella.

Polysaccharides play critical roles in bacteria, including the formation of protective capsules and biofilms and establishing specific host cell interactions. Their transport across membranes is often mediated by ATP-binding cassette (ABC) transporters, which utilize ATP to translocate diverse molecules. Cyclic ß-glucans (CßGs) are critical for host interaction of the Rhizobiales, including the zoonotic pathogen Brucella. CßGs are exported into the periplasmic space by the cyclic glucan transporter (Cgt). The interaction of an ABC transporter with a polysaccharide substrate has not been visualized so far. Here we use single-particle cryoelectron microscopy to elucidate the structures of Cgt from Brucella abortus in four conformational states. The substrate-bound structure reveals an unusual binding pocket at the height of the cytoplasmic leaflet, whereas ADP-vanadate models hint at an alternative mechanism of substrate release. Our work provides insights into the translocation of large, heterogeneous substrates and sheds light on protein-polysaccharide interactions in general.

Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.

Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.

Patient-derived monoclonal antibody neutralizes SARS-CoV-2 Omicron variants and confers full protection in monkeys.

The SARS-CoV-2 Omicron variant has very high levels of transmission, is resistant to neutralization by authorized therapeutic human monoclonal antibodies (mAb) and is less sensitive to vaccine-mediated immunity. To provide additional therapies against Omicron, we isolated a mAb named P2G3 from a previously infected vaccinated donor and showed that it has picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other variants tested. We solved the structure of P2G3 Fab in complex with the Omicron spike using cryo-electron microscopy at 3.04 Å resolution to identify the P2G3 epitope as a Class 3 mAb that is different from mAb-binding spike epitopes reported previously. Using a SARS-CoV-2 Omicron monkey challenge model, we show that P2G3 alone, or in combination with P5C3 (a broadly active Class 1 mAb previously identified), confers complete prophylactic or therapeutic protection. Although we could select for SARS-CoV-2 mutants escaping neutralization by P2G3 or by P5C3 in vitro, they had low infectivity and 'escape' mutations are extremely rare in public sequence databases. We conclude that this combination of mAbs has potential as an anti-Omicron drug.

Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation.

Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Detailed insights into the architecture of LptDE transporter complexes have been derived from X-ray crystallography. However, no structure of a laterally open LptD transporter, a transient state that occurs during LPS release, is available to date. Here, we report a cryo-EM structure of a partially opened LptDE transporter in complex with rigid chaperones derived from nanobodies, at 3.4 Å resolution. In addition, a subset of particles allows to model a structure of a laterally fully opened LptDE complex. Our work offers insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a highly rigid chaperone scaffold to enable structural biology of challenging protein targets.

Structural and molecular determinants for the interaction of ExbB from Serratia marcescens and HasB, a TonB paralog.

ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbB's are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB.

2.4-Å structure of the double-ring Gemmatimonas phototrophica photosystem.

Phototrophic Gemmatimonadetes evolved the ability to use solar energy following horizontal transfer of photosynthesis-related genes from an ancient phototrophic proteobacterium. The electron cryo-microscopy structure of the Gemmatimonas phototrophica photosystem at 2.4 Å reveals a unique, double-ring complex. Two unique membrane-extrinsic polypeptides, RC-S and RC-U, hold the central type 2 reaction center (RC) within an inner 16-subunit light-harvesting 1 (LH1) ring, which is encircled by an outer 24-subunit antenna ring (LHh) that adds light-gathering capacity. Femtosecond kinetics reveal the flow of energy within the RC-dLH complex, from the outer LHh ring to LH1 and then to the RC. This structural and functional study shows that G. phototrophica has independently evolved its own compact, robust, and highly effective architecture for harvesting and trapping solar energy.

Trans-seeding of Alzheimer-related tau protein by a yeast prion.

Abnormal tau protein aggregates constitute a hallmark of Alzheimer's disease. The mechanisms underlying the initiation of tau aggregation in sporadic neurodegeneration remain unclear. Here we investigate whether a non-human prion can seed tau aggregation. Due to their structural similarity with tau aggregates, we chose Sup35NM yeast prion domain fibrils for explorative tau seedings. Upon in vitro incubation with tau monomers, Sup35NM fibrils promoted the formation of morphologically distinct tau fibril strains. In vivo, intrahippocampal inoculation of Sup35NM fibrils accentuated tau pathology in P301S tau transgenic mice. Thus, our results provide first in vivo evidence for heterotypic cross-species seeding of a neurodegenerative human prion-like protein by a yeast prion. This opens up the conceptual perspective that non-mammalian prions present in the human microbiome could be involved in the initiation of protein misfolding in neurodegenerative disorders, a mechanism for which we propose the term "trans-seeding."

Cryo-EM structure of native human thyroglobulin.

The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its architecture is conserved among vertebrates. Synthesis of triiodothyronine (T3) and thyroxine (T4) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography-mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations.