Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. Here we develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and use solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise a mixture of single protofilament and two protofilament fibrils with very low twist. The protofilament fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural characterization of LBD Asyn fibrils and approaches for studying disease mechanisms, imaging agents and therapeutics targeting Asyn.

Apolipoprotein E secreted by astrocytes forms antiparallel dimers in discoidal lipoproteins.

The Apolipoprotein E gene (APOE) is of great interest due to its role as a risk factor for late-onset Alzheimer's disease. ApoE is secreted by astrocytes in the central nervous system in high-density lipoprotein (HDL)-like lipoproteins. Structural models of lipidated ApoE of high resolution could aid in a mechanistic understanding of how ApoE functions in health and disease. Using monoclonal Fab and F(ab')2 fragments, we characterize the structure of lipidated ApoE on astrocyte-secreted lipoproteins. Our results provide support for the "double-belt" model of ApoE in nascent discoidal HDL-like lipoproteins, where two ApoE proteins wrap around the nanodisc in an antiparallel conformation. We further show that lipidated, recombinant ApoE accurately models astrocyte-secreted ApoE lipoproteins. Cryogenic electron microscopy of recombinant lipidated ApoE further supports ApoE adopting antiparallel dimers in nascent discoidal lipoproteins.

Structural architecture of the acidic region of the B domain of coagulation factor V.

BACKGROUND: Coagulation factor (F)V features an A1-A2-B-A3-C1-C2 domain organization and functions as the inactive precursor of FVa, a component of the prothrombinase complex required for rapid thrombin generation in the penultimate step of the coagulation cascade. An intramolecular interaction within the large B domain (residues 710-1545) involves the basic region (BR, residues 963-1008) and acidic region (AR, residues 1493-1537) and locks FV in its inactive state. However, structural information on this important regulatory interaction or on the separate architecture of the AR and BR remains elusive due to conformational disorder of the B domain. OBJECTIVES: To reveal the structure of the BR-AR interaction or of its separate components. METHODS: The structure of FV is solved by cryogenic electron microscopy. RESULTS: A new 3.05 Å resolution cryogenic electron microscopy structure of FV confirms the overall organization of the A and C domains but resolves the segment 1507 to 1545 within a largely disordered B domain. The segment contains most of the AR and is organized as recently reported in FV short, a spliced variant of FV with a significantly shorter and less disordered B domain. CONCLUSION: The similar architecture of the AR in FV and FV short provides structural context for physiologically important interactions of this region with the BR in FV and with the basic C-terminal end of tissue factor pathway inhibitor α in FV short.

Vertebrate-class-specific binding modes of the alphavirus receptor MXRA8.

MXRA8 is a receptor for chikungunya (CHIKV) and other arthritogenic alphaviruses with mammalian hosts. However, mammalian MXRA8 does not bind to alphaviruses that infect humans and have avian reservoirs. Here, we show that avian, but not mammalian, MXRA8 can act as a receptor for Sindbis, western equine encephalitis (WEEV), and related alphaviruses with avian reservoirs. Structural analysis of duck MXRA8 complexed with WEEV reveals an inverted binding mode compared with mammalian MXRA8 bound to CHIKV. Whereas both domains of mammalian MXRA8 bind CHIKV E1 and E2, only domain 1 of avian MXRA8 engages WEEV E1, and no appreciable contacts are made with WEEV E2. Using these results, we generated a chimeric avian-mammalian MXRA8 decoy-receptor that neutralizes infection of multiple alphaviruses from distinct antigenic groups in vitro and in vivo. Thus, different alphaviruses can bind MXRA8 encoded by different vertebrate classes with distinct engagement modes, which enables development of broad-spectrum inhibitors.

Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes.

Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.

Structural analyses of ß2-glycoprotein I: is there a circular conformation?

BACKGROUND: Antiphospholipid antibodies targeting ß2-glycoprotein I (ß2GPI) cause thrombosis and pregnancy morbidity in antiphospholipid syndrome (APS) patients. How these antibodies recognize ß2GPI remains controversial. OBJECTIVES: This study aimed to elucidate the structure of ß2GPI and evaluate how pathogenic anti-domain I (DI) antibodies recognize it in human plasma. METHODS: ß2GPI was made recombinant and purified from human plasma using different protocols. Structural and functional analyses were conducted using orthogonal techniques, namely, electron microscopy, size-exclusion chromatography, single-molecule Förster resonance energy transfer, and microfluidic diffusional sizing. RESULTS: Electron microscopy and size-exclusion chromatography showed that the structure of ß2GPI produced recombinantly and purified from plasma is elongated, even when subjected to conditions previously reported to favor circularization. Single-molecule Förster resonance energy transfer analyses of ß2GPI labeled at positions 88 in DII and 278 in DV showed that these residues are located >90 Å apart, consistent with an elongated form. They also documented that the distance between these 2 residues did not change when the protein was reconstituted in human plasma. Microfluidic diffusional sizing documented that ß2GPI binds with moderate affinity to a prototypical anti-DI antibody targeting the epitope G40-R43 despite being elongated. CONCLUSION: Circulating ß2GPI is elongated and, therefore, fully capable of binding to anti-DI antibodies. Binding of ß2GPI to negatively charged phospholipids drives autoantibody recognition by increasing the local concentration of the antigen and not by dramatically changing its conformation. These findings clarify the structural properties of ß2GPI, which have important implications for understanding APS pathogenesis and the development of APS diagnostics and therapeutics.

Homodecameric Rad52 promotes single-position Rad51 nucleation in homologous recombination.

Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Saccharomyces cerevisiae Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, in vitro, Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, in vivo, Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR.

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.

Structure of Venezuelan equine encephalitis virus in complex with the LDLRAD3 receptor.

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)1, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus-receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.

The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies.

Although neutralizing monoclonal antibodies (mAbs) against epitopes within the alphavirus E2 protein can protect against infection, the functional significance of non-neutralizing mAbs is poorly understood. Here, we evaluate the activity of 13 non-neutralizing mAbs against Mayaro virus (MAYV), an emerging arthritogenic alphavirus. These mAbs bind to the MAYV virion and surface of infected cells but fail to neutralize infection in cell culture. Mapping studies identify six mAb binding groups that localize to discrete epitopes within or adjacent to the A domain of the E2 glycoprotein. Remarkably, passive transfer of non-neutralizing mAbs protects against MAYV infection and disease in mice, and their efficacy requires Fc effector functions. Monocytes mediate the protection of non-neutralizing mAbs in vivo, as Fcγ-receptor-expressing myeloid cells facilitate the binding, uptake, and clearance of MAYV without antibody-dependent enhancement of infection. Humoral protection against alphaviruses likely reflects contributions from non-neutralizing antibodies through Fc-dependent mechanisms that accelerate viral clearance.

LDLRAD3 is a receptor for Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans1. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)-a highly conserved yet poorly characterized member of the scavenger receptor superfamily-as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.

A molecular understanding of alphavirus entry.

Alphaviruses cause severe human illnesses including persistent arthritis and fatal encephalitis. As alphavirus entry into target cells is the first step in infection, intensive research efforts have focused on elucidating aspects of this pathway, including attachment, internalization, and fusion. Herein, we review recent developments in the molecular understanding of alphavirus entry both in vitro and in vivo and how these advances might enable the design of therapeutics targeting this critical step in the alphavirus life cycle.

An Evolutionary Insertion in the Mxra8 Receptor-Binding Site Confers Resistance to Alphavirus Infection and Pathogenesis.

Alphaviruses are emerging, mosquito-transmitted RNA viruses with poorly understood cellular tropism and species selectivity. Mxra8 is a receptor for multiple alphaviruses including chikungunya virus (CHIKV). We discovered that while expression of mouse, rat, chimpanzee, dog, horse, goat, sheep, and human Mxra8 enables alphavirus infection in cell culture, cattle Mxra8 does not. Cattle Mxra8 encodes a 15-amino acid insertion in its ectodomain that prevents Mxra8 binding to CHIKV. Identical insertions are present in zebu, yak, and the extinct auroch. As other Bovinae lineages contain related Mxra8 sequences, this insertion likely occurred at least 5 million years ago. Removing the Mxra8 insertion in Bovinae enhances alphavirus binding and infection, while introducing the insertion into mouse Mxra8 blocks CHIKV binding, prevents infection by multiple alphaviruses in cells, and mitigates CHIKV-induced pathogenesis in mice. Our studies on how this insertion provides resistance to CHIKV infection could facilitate countermeasures that disrupt Mxra8 interactions with alphaviruses.

Neutralizing antibodies against Mayaro virus require Fc effector functions for protective activity.

Despite causing outbreaks of fever and arthritis in multiple countries, no countermeasures exist against Mayaro virus (MAYV), an emerging mosquito-transmitted alphavirus. We generated 18 neutralizing mAbs against MAYV, 11 of which had "elite" activity that inhibited infection with EC50 values of <10 ng/ml. Antibodies with the greatest inhibitory capacity in cell culture mapped to epitopes near the fusion peptide of E1 and in domain B of the E2 glycoproteins. Unexpectedly, many of the elite neutralizing mAbs failed to prevent MAYV infection and disease in vivo. Instead, the most protective mAbs bound viral antigen on the cell surface with high avidity and promoted specific Fc effector functions, including phagocytosis by neutrophils and monocytes. In subclass switching studies, murine IgG2a and humanized IgG1 mAb variants controlled infection better than murine IgG1 and humanized IgG1-N297Q variants. An optimally protective antibody response to MAYV and possibly other alphaviruses may require tandem virus neutralization by the Fab moiety and effector functions of the Fc region.

Cryo-EM Structure of Chikungunya Virus in Complex with the Mxra8 Receptor.

Mxra8 is a receptor for multiple arthritogenic alphaviruses that cause debilitating acute and chronic musculoskeletal disease in humans. Herein, we present a 2.2 Å resolution X-ray crystal structure of Mxra8 and 4 to 5 Å resolution cryo-electron microscopy reconstructions of Mxra8 bound to chikungunya (CHIKV) virus-like particles and infectious virus. The Mxra8 ectodomain contains two strand-swapped Ig-like domains oriented in a unique disulfide-linked head-to-head arrangement. Mxra8 binds by wedging into a cleft created by two adjacent CHIKV E2-E1 heterodimers in one trimeric spike and engaging a neighboring spike. Two binding modes are observed with the fully mature VLP, with one Mxra8 binding with unique contacts. Only the high-affinity binding mode was observed in the complex with infectious CHIKV, as viral maturation and E3 occupancy appear to influence receptor binding-site usage. Our studies provide insight into how Mxra8 binds CHIKV and creates a path for developing alphavirus entry inhibitors.

Protective antibodies against Eastern equine encephalitis virus bind to epitopes in domains A and B of the E2 glycoprotein.

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have 'elite' activity with 50 and 99% effective inhibitory concentrations (EC50 and EC99) of less than 10 and 100 ng ml-1, respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design.

Mxra8 is a receptor for multiple arthritogenic alphaviruses.

Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease 1 . The host factors required for alphavirus entry remain poorly characterized 2 . Here we use a genome-wide CRISPR-Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O'nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection. Mxra8 bound directly to chikungunya virus particles and enhanced virus attachment and internalization into cells. Consistent with these findings, Mxra8-Fc fusion protein or anti-Mxra8 monoclonal antibodies blocked chikungunya virus infection in multiple cell types, including primary human synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells. Mutagenesis experiments suggest that Mxra8 binds to a surface-exposed region across the A and B domains of chikungunya virus E2 protein, which are a speculated site of attachment. Finally, administration of the Mxra8-Fc protein or anti-Mxra8 blocking antibodies to mice reduced chikungunya and O'nyong nyong virus infection as well as associated foot swelling. Pharmacological targeting of Mxra8 could form a strategy for mitigating infection and disease by multiple arthritogenic alphaviruses.

An upstream open reading frame (uORF) signals for cellular localization of the virulence factor implicated in pregnancy associated malaria.

Plasmodium falciparum, the causative agent of the deadliest form of human malaria, alternates expression of variable antigens, encoded by members of a multi-copy gene family named var. In var2csa, the var gene implicated in pregnancy-associated malaria, translational repression is regulated by a unique upstream open reading frame (uORF) found only in its 5' UTR. Here, we report that this translated uORF significantly alters both transcription and posttranslational protein trafficking. The parasite can alter a protein's destination without any modifications to the protein itself, but instead by an element within the 5' UTR of the transcript. This uORF-dependent localization was confirmed by single molecule STORM imaging, followed by fusion of the uORF to a reporter gene which changes its cellular localization from cytoplasmic to ER-associated. These data point towards a novel regulatory role of uORF in protein trafficking, with important implications for the pathology of pregnancy-associated malaria.

Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors.

Malaria parasites increase their host erythrocyte's permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against in vitro parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel's structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing.

How do antimalarial drugs reach their intracellular targets?

Drugs represent the primary treatment available for human malaria, as caused by Plasmodium spp. Currently approved drugs and antimalarial drug leads generally work against parasite enzymes or activities within infected erythrocytes. To reach their specific targets, these chemicals must cross at least three membranes beginning with the host cell membrane. Uptake at each membrane may involve partitioning and diffusion through the lipid bilayer or facilitated transport through channels or carriers. Here, we review the features of available antimalarials and examine whether transporters may be required for their uptake. Our computational analysis suggests that most antimalarials have high intrinsic membrane permeability, obviating the need for uptake via transporters; a subset of compounds appear to require facilitated uptake. We also review parasite and host transporters that may contribute to drug uptake. Broad permeability channels at the erythrocyte and parasitophorous vacuolar membranes of infected cells relax permeability constraints on antimalarial drug design; however, this uptake mechanism is prone to acquired resistance as the parasite may alter channel activity to reduce drug uptake. A better understanding of how antimalarial drugs reach their intracellular targets is critical to prioritizing drug leads for antimalarial development and may reveal new targets for therapeutic intervention.