ABSTRACT
Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease.
Subject(s)
Amyloid , Islet Amyloid Polypeptide , Humans , Amyloid/chemistry , Cryoelectron Microscopy , Islet Amyloid Polypeptide/chemistry , Amyloidogenic ProteinsABSTRACT
The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-ß structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention.
Subject(s)
Amyloid/metabolism , Amyloid/physiology , Amyloid/ultrastructure , Alzheimer Disease/physiopathology , Amyloidosis/physiopathology , Diabetes Mellitus, Type 2/physiopathology , Humans , Parkinson Disease/physiopathology , Plaque, Amyloid/metabolism , Plaque, Amyloid/physiopathologyABSTRACT
A defining pathological feature of most neurodegenerative diseases is the assembly of proteins into amyloid that form disease-specific structures1. In Alzheimer's disease, this is characterized by the deposition of ß-amyloid and tau with disease-specific conformations. The in situ structure of amyloid in the human brain is unknown. Here, using cryo-fluorescence microscopy-targeted cryo-sectioning, cryo-focused ion beam-scanning electron microscopy lift-out and cryo-electron tomography, we determined in-tissue architectures of ß-amyloid and tau pathology in a postmortem Alzheimer's disease donor brain. ß-amyloid plaques contained a mixture of fibrils, some of which were branched, and protofilaments, arranged in parallel arrays and lattice-like structures. Extracellular vesicles and cuboidal particles defined the non-amyloid constituents of ß-amyloid plaques. By contrast, tau inclusions formed parallel clusters of unbranched filaments. Subtomogram averaging a cluster of 136 tau filaments in a single tomogram revealed the polypeptide backbone conformation and filament polarity orientation of paired helical filaments within tissue. Filaments within most clusters were similar to each other, but were different between clusters, showing amyloid heterogeneity that is spatially organized by subcellular location. The in situ structural approaches outlined here for human donor tissues have applications to a broad range of neurodegenerative diseases.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Brain , Cryoelectron Microscopy , Electron Microscope Tomography , Plaque, Amyloid , tau Proteins , Humans , Male , Mice , Alzheimer Disease/pathology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/ultrastructure , Autopsy , Brain/metabolism , Brain/pathology , Brain/ultrastructure , Extracellular Vesicles/metabolism , Extracellular Vesicles/chemistry , Extracellular Vesicles/ultrastructure , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Plaque, Amyloid/chemistry , Plaque, Amyloid/ultrastructure , tau Proteins/chemistry , tau Proteins/metabolism , tau Proteins/ultrastructureABSTRACT
Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut1. Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome.
Subject(s)
Bacterial Outer Membrane Proteins , Bacterial Outer Membrane , Bacteroides thetaiotaomicron , Gastrointestinal Tract , Polysaccharides , Humans , Bacterial Outer Membrane/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacteroides thetaiotaomicron/enzymology , Bacteroides thetaiotaomicron/metabolism , Gastrointestinal Tract/metabolism , Gastrointestinal Tract/microbiology , Glycoside Hydrolases/metabolism , Polysaccharides/metabolismABSTRACT
Amyloid formation by α-synuclein (αSyn) occurs in Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Deciphering the residues that regulate αSyn amyloid fibril formation will not only provide mechanistic insight but may also reveal targets to prevent and treat disease. Previous investigations have identified several regions of αSyn to be important in the regulation of amyloid formation, including the non-amyloid-ß component (NAC), P1 region (residues 36 to 42), and residues in the C-terminal domain. Recent studies have also indicated the importance of the N-terminal region of αSyn for both its physiological and pathological roles. Here, the role of residues 2 to 7 in the N-terminal region of αSyn is investigated in terms of their ability to regulate amyloid fibril formation in vitro and in vivo. Deletion of these residues (αSynΔN7) slows the rate of fibril formation in vitro and reduces the capacity of the protein to be recruited by wild-type (αSynWT) fibril seeds, despite cryo-EM showing a fibril structure consistent with those of full-length αSyn. Strikingly, fibril formation of αSynΔN7 is not induced by liposomes, despite the protein binding to liposomes with similar affinity to αSynWT. A Caenorhabditis elegans model also showed that αSynΔN7::YFP forms few puncta and lacks motility and lifespan defects typified by expression of αSynWT::YFP. Together, the results demonstrate the involvement of residues 2 to 7 of αSyn in amyloid formation, revealing a target for the design of amyloid inhibitors that may leave the functional role of the protein in membrane binding unperturbed.
Subject(s)
Amyloid , Caenorhabditis elegans , alpha-Synuclein , alpha-Synuclein/metabolism , alpha-Synuclein/genetics , alpha-Synuclein/chemistry , Amyloid/metabolism , Caenorhabditis elegans/metabolism , Animals , Humans , Lipids/chemistry , Parkinson Disease/metabolism , Parkinson Disease/genetics , Parkinson Disease/pathologyABSTRACT
α-, ß-, and γ-Synuclein are intrinsically disordered proteins implicated in physiological processes in the nervous system of vertebrates. α-synuclein (αSyn) is the amyloidogenic protein associated with Parkinson's disease and certain other neurodegenerative disorders. Intensive research has focused on the mechanisms that cause αSyn to form amyloid structures, identifying its NAC region as being necessary and sufficient for amyloid assembly. Recent work has shown that a 7-residue sequence (P1) is necessary for αSyn amyloid formation. Although γ-synuclein (γSyn) is 55% identical in sequence to αSyn and its pathological deposits are also observed in association with neurodegenerative conditions, γSyn is resilient to amyloid formation in vitro. Here, we report a rare single nucleotide polymorphism (SNP) in the SNCG gene encoding γSyn, found in two patients with amyotrophic lateral sclerosis (ALS). The SNP results in the substitution of Met38 with Ile in the P1 region of the protein. These individuals also had a second, common and nonpathological, SNP in SNCG resulting in the substitution of Glu110 with Val. In vitro studies demonstrate that the Ile38 variant accelerates amyloid fibril assembly. Contrastingly, Val110 retards fibril assembly and mitigates the effect of Ile38. Substitution of residue 38 with Leu had little effect, while Val retards, and Ala increases the rate of amyloid formation. Ile38 γSyn also results in the formation of γSyn-containing inclusions in cells. The results show how a single point substitution can enhance amyloid formation of γSyn and highlight the P1 region in driving amyloid formation in another synuclein family member.
Subject(s)
Amyotrophic Lateral Sclerosis , Parkinson Disease , Animals , Humans , Amyloid/chemistry , Amyotrophic Lateral Sclerosis/genetics , gamma-Synuclein/genetics , alpha-Synuclein/metabolism , Parkinson Disease/metabolism , Amyloidogenic ProteinsABSTRACT
Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads1, which prevents filament formation and inactivates the motors2. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations.
Subject(s)
Cryoelectron Microscopy , Myosin Type II/chemistry , Myosin Type II/ultrastructure , Animals , Enzyme Activation , Enzyme Stability , Models, Molecular , Muscle, Smooth/chemistry , Myosin Light Chains/chemistry , Myosin Light Chains/metabolism , Myosin Light Chains/ultrastructure , Myosin Type II/metabolism , Phosphorylation , Protein Domains , TurkeysABSTRACT
The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1-3. Electron transfer within cytb6 fâ occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 fâ also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 fâ monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 fâ structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.
Subject(s)
Cryoelectron Microscopy , Cytochrome b6f Complex/chemistry , Cytochrome b6f Complex/ultrastructure , Spinacia oleracea/chemistry , Spinacia oleracea/ultrastructure , Binding Sites , Chlorophyll/chemistry , Heme/chemistry , Lipids/chemistry , Models, Molecular , Oxidation-Reduction , Photosynthesis , Plastoquinone/chemistry , Structure-Activity RelationshipABSTRACT
Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC-SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer-and not the active PLP-bound tetramer-binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling.
Subject(s)
Deubiquitinating Enzymes/metabolism , Glycine Hydroxymethyltransferase/metabolism , Interferon Type I/immunology , Multienzyme Complexes/immunology , Multienzyme Complexes/metabolism , Signal Transduction/immunology , Cryoelectron Microscopy , Deubiquitinating Enzymes/antagonists & inhibitors , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/ultrastructure , Glycine Hydroxymethyltransferase/ultrastructure , HEK293 Cells , Humans , Inflammation/immunology , Models, Molecular , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Mutation , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Pyridoxal Phosphate/metabolismABSTRACT
The first member of the pleuromutilin (PLM) class suitable for systemic antibacterial chemotherapy in humans recently entered clinical use, underscoring the need to better understand mechanisms of PLM resistance in disease-causing bacterial genera. Of the proteins reported to mediate PLM resistance in staphylococci, the least-well studied to date is Sal(A), a putative ABC-F NTPase that-by analogy to other proteins of this type-may act to protect the ribosome from PLMs. Here, we establish the importance of Sal proteins as a common source of PLM resistance across multiple species of staphylococci. Sal(A) is revealed as but one member of a larger group of Sal-type ABC-F proteins that vary considerably in their ability to mediate resistance to PLMs and other antibiotics. We find that specific sal genes are intrinsic to particular staphylococcal species, and show that this gene family is likely ancestral to the genus Staphylococcus. Finally, we solve the cryo-EM structure of a representative Sal-type protein (Sal(B)) in complex with the staphylococcal 70S ribosome, revealing that Sal-type proteins bind into the E site to mediate target protection, likely by displacing PLMs and other antibiotics via an allosteric mechanism.
Subject(s)
Diterpenes , Polycyclic Compounds , Anti-Bacterial Agents/chemistry , Bacterial Proteins/metabolism , Diterpenes/pharmacology , Humans , Polycyclic Compounds/pharmacology , Staphylococcus/genetics , Staphylococcus/metabolism , PleuromutilinsABSTRACT
Icosahedral viral capsids must undergo conformational rearrangements to coordinate essential processes during the viral life cycle. Capturing such conformational flexibility has been technically challenging yet could be key for developing rational therapeutic agents to combat infections. Noroviruses are nonenveloped, icosahedral viruses of global importance to human health. They are a common cause of acute gastroenteritis, yet no vaccines or specific antiviral agents are available. Here, we use genetics and cryo-electron microscopy (cryo-EM) to study the high-resolution solution structures of murine norovirus as a model for human viruses. By comparing our 3 structures (at 2.9- to 3.1-Å resolution), we show that whilst there is little change to the shell domain of the capsid, the radiating protruding domains are flexible, adopting distinct states both independently and synchronously. In doing so, the capsids sample a range of conformational space, with implications for maintaining virion stability and infectivity.
Subject(s)
Capsid/chemistry , Norovirus/chemistry , Animals , Capsid Proteins/chemistry , Capsid Proteins/genetics , Cryoelectron Microscopy , Dimerization , Hot Temperature , Mice , Models, Molecular , Mutation , Norovirus/genetics , Norovirus/pathogenicity , Protein Domains , RAW 264.7 Cells , Structure-Activity RelationshipABSTRACT
The ß-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E.â coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Electron Spin Resonance Spectroscopy , Protein FoldingABSTRACT
Picornaviruses are important viral pathogens, but despite extensive study, the assembly process of their infectious virions is still incompletely understood, preventing the development of anti-viral strategies targeting this essential part of the life cycle. We report the identification, via RNA SELEX and bioinformatics, of multiple RNA sites across the genome of a typical enterovirus, enterovirus-E (EV-E), that each have affinity for the cognate viral capsid protein (CP) capsomer. Many of these sites are evolutionarily conserved across known EV-E variants, suggesting they play essential functional roles. Cryo-electron microscopy was used to reconstruct the EV-E particle at ~2.2 Å resolution, revealing extensive density for the genomic RNA. Relaxing the imposed symmetry within the reconstructed particles reveals multiple RNA-CP contacts, a first for any picornavirus. Conservative mutagenesis of the individual RNA-contacting amino acid side chains in EV-E, many of which are conserved across the enterovirus family including poliovirus, is lethal but does not interfere with replication or translation. Anti-EV-E and anti-poliovirus aptamers share sequence similarities with sites distributed across the poliovirus genome. These data are consistent with the hypothesis that these RNA-CP contacts are RNA Packaging Signals (PSs) that play vital roles in assembly and suggest that the RNA PSs are evolutionarily conserved between pathogens within the family, augmenting the current protein-only assembly paradigm for this family of viruses.
Subject(s)
Capsid Proteins/metabolism , Enterovirus/physiology , RNA, Viral/genetics , Virus Assembly/physiology , Amino Acid Sequence , Capsid Proteins/ultrastructure , Cryoelectron Microscopy , Enterovirus/ultrastructure , RNA, Viral/ultrastructureABSTRACT
Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit ß-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.
Subject(s)
Bacteriophages/ultrastructure , Capsid/ultrastructure , DNA Packaging/genetics , DNA Viruses/ultrastructure , Bacteriophages/genetics , Cryoelectron Microscopy , DNA Viruses/genetics , DNA, Viral/genetics , DNA, Viral/ultrastructure , Virion/genetics , Virion/ultrastructure , Virus Assembly/geneticsABSTRACT
DNA nanotechnology allows for the design of programmable DNA-built nanodevices which controllably interact with biological membranes and even mimic the function of natural membrane proteins. Hydrophobic modifications, covalently linked to the DNA, are essential for targeted interfacing of DNA nanostructures with lipid membranes. However, these hydrophobic tags typically induce undesired aggregation eliminating structural control, the primary advantage of DNA nanotechnology. Here, we study the aggregation of cholesterol-modified DNA nanostructures using a combined approach of non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering, confocal microscopy and atomistic molecular dynamics simulations. We show that the aggregation of cholesterol-tagged ssDNA is sequence-dependent, while for assembled DNA constructs, the number and position of the cholesterol tags are the dominating factors. Molecular dynamics simulations of cholesterol-modified ssDNA reveal that the nucleotides wrap around the hydrophobic moiety, shielding it from the environment. Utilizing this behavior, we demonstrate experimentally that the aggregation of cholesterol-modified DNA nanostructures can be controlled by the length of ssDNA overhangs positioned adjacent to the cholesterol. Our easy-to-implement method for tuning cholesterol-mediated aggregation allows for increased control and a closer structure-function relationship of membrane-interfacing DNA constructs - a fundamental prerequisite for employing DNA nanodevices in research and biomedicine.
Subject(s)
Chemical Precipitation , Cholesterol/chemistry , DNA, Single-Stranded , Nanostructures/chemistry , Nanotechnology/methods , Base Sequence/physiology , Cell Membrane/chemistry , Cell Membrane/metabolism , Cholesterol/metabolism , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Molecular Docking Simulation , Nucleic Acid ConformationABSTRACT
Acute hemorrhagic conjunctivitis (AHC) is a painful, contagious eye disease, with millions of cases in the last decades. Coxsackievirus A24 (CV-A24) was not originally associated with human disease, but in 1970 a pathogenic "variant" (CV-A24v) emerged, which is now the main cause of AHC. Initially, this variant circulated only in Southeast Asia, but it later spread worldwide, accounting for numerous AHC outbreaks and two pandemics. While both CV-A24 variant and nonvariant strains still circulate in humans, only variant strains cause AHC for reasons that are yet unknown. Since receptors are important determinants of viral tropism, we set out to map the CV-A24 receptor repertoire and establish whether changes in receptor preference have led to the increased pathogenicity and rapid spread of CV-A24v. Here, we identify ICAM-1 as an essential receptor for both AHC-causing and non-AHC strains. We provide a high-resolution cryo-EM structure of a virus-ICAM-1 complex, which revealed critical ICAM-1-binding residues. These data could help identify a possible conserved mode of receptor engagement among ICAM-1-binding enteroviruses and rhinoviruses. Moreover, we identify a single capsid substitution that has been adopted by all pandemic CV-A24v strains and we reveal that this adaptation enhances the capacity of CV-A24v to bind sialic acid. Our data elucidate the CV-A24v receptor repertoire and point to a role of enhanced receptor engagement in the adaptation to the eye, possibly enabling pandemic spread.
Subject(s)
Conjunctivitis, Acute Hemorrhagic/metabolism , Enterovirus C, Human/metabolism , Intercellular Adhesion Molecule-1/metabolism , Receptors, Virus/metabolism , Amino Acid Sequence , Capsid Proteins/genetics , Capsid Proteins/metabolism , Conjunctivitis, Acute Hemorrhagic/epidemiology , Conjunctivitis, Acute Hemorrhagic/virology , Cryoelectron Microscopy , Disease Outbreaks , Enterovirus C, Human/genetics , Enterovirus C, Human/physiology , Humans , Intercellular Adhesion Molecule-1/chemistry , Mutation , N-Acetylneuraminic Acid/metabolism , Pandemics , Phylogeny , Protein Binding , Receptors, Virus/chemistry , Sequence Homology, Amino Acid , Viral Tropism/physiologyABSTRACT
Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.
Subject(s)
Antiviral Agents/pharmacology , Coxsackievirus Infections/diet therapy , Enterovirus C, Human/drug effects , N-Acetylneuraminic Acid/pharmacology , Conjunctivitis, Acute Hemorrhagic/drug therapy , Conjunctivitis, Acute Hemorrhagic/metabolism , Conjunctivitis, Acute Hemorrhagic/virology , Coxsackievirus Infections/metabolism , Coxsackievirus Infections/virology , Glucose/metabolism , Humans , Lectins/metabolism , Ligands , Polysaccharides/metabolism , Receptors, Virus/metabolismABSTRACT
Improvements in technology often drive scientific discovery. Therefore, research requires sustained investment in the latest equipment and training for the researchers who are going to use it. Prioritising and administering infrastructure investment is challenging because future needs are difficult to predict. In the past, highly computationally demanding research was associated primarily with particle physics and astronomy experiments. However, as biology becomes more quantitative and bioscientists generate more and more data, their computational requirements may ultimately exceed those of physical scientists. Computation has always been central to bioinformatics, but now imaging experiments have rapidly growing data processing and storage requirements. There is also an urgent need for new modelling and simulation tools to provide insight and understanding of these biophysical experiments. Bioscience communities must work together to provide the software and skills training needed in their areas. Research-active institutions need to recognise that computation is now vital in many more areas of discovery and create an environment where it can be embraced. The public must also become aware of both the power and limitations of computing, particularly with respect to their health and personal data.
Subject(s)
Computational Biology/trends , Data Curation/trends , Animals , Computer Simulation/trends , Humans , Models, Biological , SoftwareABSTRACT
Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using ß2-microglobulin (ß2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.
Subject(s)
Amyloid/chemistry , Amyloidosis/metabolism , Benzothiazoles , Endosomes/chemistry , HSP70 Heat-Shock Proteins/chemistry , Humans , Hydrogen-Ion Concentration , Kinetics , Lysosomes/chemistry , Monocytes/metabolism , Muramidase/chemistry , Protein Binding , Recombinant Proteins/chemistry , Spectrometry, Fluorescence , Thiazoles/chemistry , beta 2-Microglobulin/chemistryABSTRACT
BK polyomavirus (BKPyV; hereafter referred to as BK) causes a lifelong chronic infection and is associated with debilitating disease in kidney transplant recipients. Despite its importance, aspects of the virus life cycle remain poorly understood. In addition to the structural proteins, the late region of the BK genome encodes for an auxiliary protein called agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to virion infectivity. Here, we demonstrate an essential role for agnoprotein in BK virus release. Viruses lacking agnoprotein fail to release from host cells and do not propagate to wild-type levels. Despite this, agnoprotein is not essential for virion infectivity or morphogenesis. Instead, agnoprotein expression correlates with nuclear egress of BK virions. We demonstrate that the agnoprotein binding partner α-soluble N-ethylmaleimide sensitive fusion (NSF) attachment protein (α-SNAP) is necessary for BK virion release, and siRNA knockdown of α-SNAP prevents nuclear release of wild-type BK virions. These data highlight a novel role for agnoprotein and begin to reveal the mechanism by which polyomaviruses leave an infected cell.