Negative Stain Electron Microscopy Research Articles

A multispecific antibody against SARS-CoV-2 prevents immune escape in vitro and confers prophylactic protection in vivo.

Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trivalent trispecific antibody that potently neutralized diverse SARS-CoV-2 variants and prevented virus escape more effectively than single antibodies or mixtures of the parental antibodies. Despite being generated before the appearance of Omicron, this trispecific antibody neutralized all major Omicron variants through BA.4/BA.5 at nanomolar concentrations. Negative stain electron microscopy suggested that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated binding across more than one spike protein. Moreover, a tetravalent trispecific antibody containing the same variable regions as the trivalent trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2, and BA.5 challenge, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. These results demonstrated that multispecific antibodies have the potential to provide broad SARS-CoV-2 coverage, decrease the likelihood of escape, simplify treatment, and provide a strategy for antibody therapies that could help eliminate pandemic spread for this and other pathogens.

7222 Structural and Mechanistic Studies of the Bioactive Anti-Müllerian Hormone Procomplex

Abstract Disclosure: J.A. Howard: None. L. Hok: None. N.J. Sanford: None. R.L. Cate: None. K. Hart: Employee; Self; Regeneron Pharmaceuticals. E. Leach: None. D. Pepin: None. P.K. Donahoe: None. T.B. Thompson: Advisory Board Member; Self; Oviva Therapeutics, Keros Therapeutics. The Anti-Mullerian hormone (AMH), a divergent member of the TGF-beta signaling superfamily, plays crucial roles in reproductive development and maintenance. Owing to its key role as a negative feedback regulator in folliculogenesis, AMH has garnered significant interest for therapeutic applications in contraception and oncofertility. The bioactive form of AMH is a noncovalent complex, comprising the TGFβ-like signaling domain and a dimeric prodomain whose structure remains largely unexplored. The prodomain component is necessary for the dimerization and secretion of the signaling domain, shuttling the ligand to autocrine, paracrine, and endocrine targets. In contrast, the structure and biochemistry of this domain remain poorly characterized, and its role in enhancing AMH signaling remains unclear. This gap in understanding significantly limits the enhancement and wider application of AMH-based protein therapeutics. Molecular modeling unveiled an unexpected AMH prodomain structure: an N-terminal dimerizing domain (NTD) linked to a novel C-terminal binding domain (CTD). This atypical design was affirmed by both small-angle X-ray scattering and negative-stain EM. The CTD introduces a unique TGFβ superfamily fold consisting of a four-helix bundle and an unstructured binding “belt”. The more prodomain-like NTD does not interact with the signaling ligand, nor does it consistently interact with the CTD. Both domains exhibit high levels of interspecies conservation as well as disease-causing mutations, such as those found in PMDS, PCOS, and POI. Using single-particle Cryo-EM we have elucidated the molecular mechanisms of the interaction between the AMH prodomain and signaling domain bound by mAb 6E11. Our 3.2Å cryo-EM structure confirmed the predicted architecture and binding function of the CTD and demonstrated a new dimension of conformational plasticity within the signaling ligand. We observed movements up to 5Å at the fingertips and 10Å in the outer helices of the CTD. More unexpectedly, the conformation of the AMH signaling ligand in the procomplex form is far more open and extended than has been observed in any other structure of a TGF-beta superfamily ligand, including the AMH ligand bound to its receptor AMHR2. We see that transitioning from a prodomain-bound to a receptor-bound state involves significant conformational remodeling of the signaling domain. This reorganization supports a conformational shift mechanism for AMH signaling in which bivalent binding of AMHR2 at the cell surface induces a >15Å and 38° closing of the ligand fingertips, leading to prodomain displacement and subsequent recruitment of type I receptors. Our findings correct earlier structural misconceptions and build a comprehensive model for interpreting existing functional data. This structure provides a foundation for understanding AMH dysregulation and developing advanced AMH therapeutics. Presentation: 6/1/2024

DNA-Based Molecular Clamp for Probing Protein Interactions and Structure under Force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology, and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA-based device in which the transition between single- and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension, while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Modular Structure and Polymerization Status of GABAA Receptors Illustrated with EM Analysis and AlphaFold2 Prediction.

Type-A γ-aminobutyric acid (GABAA) receptors are channel proteins crucial to mediating neuronal balance in the central nervous system (CNS). The structure of GABAA receptors allows for multiple binding sites and is key to drug development. Yet the formation mechanism of the receptor's distinctive pentameric structure is still unknown. This study aims to investigate the role of three predominant subunits of the human GABAA receptor in the formation of protein pentamers. Through purifying and refolding the protein fragments of the GABAA receptor α1, β2, and γ2 subunits, the particle structures were visualised with negative staining electron microscopy (EM). To aid the analysis, AlphaFold2 was used to compare the structures. Results show that α1 and β2 subunit fragments successfully formed homo-oligomers, particularly homopentameric structures, while the predominant heteropentameric GABAA receptor was also replicated through the combination of the three subunits. However, homopentameric structures were not observed with the γ2 subunit proteins. A comparison of the AlphaFold2 predictions and the previously obtained cryo-EM structures presents new insights into the subunits' modular structure and polymerization status. By performing experimental and computational studies, a deeper understanding of the complex structure of GABAA receptors is provided. Hopefully, this study can pave the way to developing novel therapeutics for neuropsychiatric diseases.

Design and immunogenicity of an HIV-1 clade C pediatric envelope glycoprotein stabilized by multiple platforms.

Various design platforms are available to stabilize soluble HIV-1 envelope (Env) trimers, which can be used as antigenic baits and vaccine antigens. However, stabilizing HIV-1 clade C trimers can be challenging. Here, we stabilized an HIV-1 clade C trimer based on an Env isolated from a pediatric elite-neutralizer (AIIMS_329) using multiple platforms, including SOSIP.v8.2, ferritin nanoparticles (NP) and an I53-50 two-component NP, followed by characterization of their biophysical, antigenic, and immunogenic properties. The stabilized 329 Envs showed binding affinity to trimer-specific HIV-1 broadly neutralizing antibodies (bnAbs), with negligible binding to non-neutralizing antibodies (non-nAbs). Negative-stain electron microscopy (nsEM) confirmed the native-like conformation of the Envs. Multimerization of 329 SOSIP.v8.2 on ferritin and two-component I53-50 NPs improved the overall affinity to HIV-1 bnAbs and immunogenicity in rabbits. These stabilized HIV-1 clade C 329 Envs demonstrate the potential to be used as antigenic baits and as components of multivalent vaccine candidates in future.

Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme binding.

Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in P. aeruginosa clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.

Unraveling the structure and function of a novel SegC protein interacting with the SegAB chromosome segregation complex in Archaea.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

Swinepox virus: an unusual outbreak in free-range pig farms in Sicily (Italy)

BackgroundTwo outbreaks of swinepox were investigated in free-range domestic pig farms located in the northeastern side of Sicily, Italy. The disease is generally self-limiting with a low mortality rate, but morbidity can reach high rates in case of poor sanitary conditions, improper husbandry practices and ectoparasitic infestation. The presented cases are the first ever reported on the island and part of the few cases reported in domestic pigs.Case presentationCarcasses condemned at the slaughterhouse and deceased pigs from Farm A and Farm B respectively, were referred for post-mortem examination and further investigations, with a strong suspect of SwinePox virus (SWPV) infection. Twelve deceased pigs were examined in total, showing poor body condition and pustular lesions scattered all over the cutaneous surfaces. Moreover, pigs from Farm B showed ocular lesions classified from Grade I to IV (from mild conjunctivitis to severe keratoconjunctivitis with corneal oedema, opacity, and ulcers). Final diagnosis was pursued by the microscopic assessment of skin lesions in both farms, which revealed the typical SWPV-lesion appearance, such as severe and disseminated ulcerative dermatitis and suspected inclusion bodies multifocally observed in the epidermis. Moreover, negative staining Electron Microscopy (nsEM) was performed on skin lesions and ocular swabs from Farm B, revealing in two samples the presence of brick-shaped viral particles, 220 nm long and 160 nm wide, with irregularly arranged surface tubules, identified as SWPV. The gene encoding the 482-bp fragment of the virus late transcription factor–3 was detected by PCR and sequencing revealed 99.79% identity and 100% query-cover with a strain previously isolated in Germany. Field clinical assessment was then performed in Farm B, revealing high overcrowding, poor sanitary conditions and improper husbandry practices, which are relevant risk factors for SWPV transmission.ConclusionsThe present is the first case report of SWPV in free-range pigs raised in Sicily, an island of the Southern coast of Italy, and wants to raise awareness on a neglected disease, and cause of animal health and welfare issues.

Formation and survival of clay-mediated protocell membranes composed of single-chain amphiphiles in aqueous solutions

Protocell compartments composed of simple amphiphilic molecules could have survived on the clay mineral surfaces which can serve well as plausible sites for prebiotic chemistry on the early Earth. Herein we chose four common single-chain amphiphiles (SCAs), including the anionic surfactant sodium dodecyl sulfate (SDS) and sodium dodecylbenzenesulfonate (SDBS), the cationic surfactant dodecyltrimethylammonium bromide (DTAB) and the zwitterionic lauryl sulfobetaine (LSB)), as model constituents of early membranes to establish a tentative model for protocell compartment. Positively charged Mg2Al-/Mg2Fe-layered double hydroxides (LDHs) and FeS2 are viewed as models of clay minerals. We demonstrate by comprehensively experimental study that unconventional vesicles can be easily promoted in a SCA aqueous micellar solution in the presence of model particles. Morphology evolutions are traced by collecting samples from mixtures of LDHs and SCA aqueous solutions and then recording at different incubation times via negative-staining or cryo-transmission electron microscopy (NS-TEM or cryo-TEM) and dynamic light scattering (DLS). Additionally, the stability of SCA bilayer membranes under possible prebiotic environment are tentatively studied. This work not only offers a robust yet versatile approach for the fabrication of novel vesicles but also provides new insights into the origin of cellular life.

In vitro higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs.

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.

Structural Mapping of Polyclonal IgG Responses to HA After Influenza Virus Vaccination or Infection.

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing context for future vaccine trials and emphasizing the importance of carefully designing vaccines to boost protective responses towards conserved epitopes.

Elasticity and Thermal Stability are Key Determinants of Hearing Rescue by Mini-Protocadherin-15 Proteins.

Protocadherin-15 is a core protein component of inner-ear hair-cell tip links pulling on transduction channels essential for hearing and balance. Protocadherin-15 defects can result in non-syndromic deafness or Usher syndrome type 1F (USH1F) with hearing loss, balance deficits, and progressive blindness. Three rationally engineered shortened versions of protocadherin-15 (mini-PCDH15s) amenable for gene therapy have been used to rescue function in USH1F mouse models. Two can successfully or partially rescue hearing, while another one fails. Here we show that despite varying levels of hearing rescue, all three mini-PCDH15 versions can rescue hair-cell mechanotransduction. Negative-stain electron microscopy shows that all three versions form dimers like the wild-type protein, while crystal structures of some engineered fragments show that these can properly fold and bind calcium ions essential for function. In contrast, simulations predict distinct elasticities and nano differential scanning fluorimetry shows differences in melting temperature measurements. Our data suggest that elasticity and thermal stability are key determinants of sustained hearing rescue by mini-PCDH15s.

Midkine Attenuates Aβ Fibril Assembly and AmyloidPlaque Formation.

Proteomic profiling of Alzheimer's disease (AD) brains has identified numerous understudied proteins, including midkine (MDK), that are highly upregulated and correlated with Aβ since the early disease stage, but their roles in disease progression are not fully understood. Here we present that MDK attenuates Aβ assembly and influences amyloid formation in the 5xFAD amyloidosis mouse model. MDK protein mitigates fibril formation of both Aβ40 and Aβ42 peptides in Thioflavin T fluorescence assay, circular dichroism, negative stain electron microscopy, and NMR analysis. Knockout of Mdkgene in 5xFAD increases amyloid formation and microglial activation. Further comprehensive mass spectrometry-based profiling of whole proteome and aggregated proteome in these mouse models indicates significant accumulation of Aβ and Aβ-correlated proteins, along with microglial components. Thus, our structural and mouse model studies reveal a protective role of MDK in counteracting amyloid pathology in Alzheimer's disease.

A DNA-based molecular clamp for probing protein interactions and structure under force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA based device in which the transition between single-stranded and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Xinyang flavivirus, from Haemaphysalis flava ticks in Henan Province, China, defines a basal, likely tick-only Orthoflavivirus clade.

Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75 % in adult females and 15.19 % in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.

Detection of macropodid alphaherpesvirus 2 infection and lesions in sudden death of a captive Virginia opossum and a water opossum.

Macropodid alphaherpesvirus 2 (MaAHV2) is best described in macropods and has been implicated in outbreaks among captive marsupial populations in Australia. Natural disease caused by herpesviruses has not been reported previously in opossum species, to our knowledge. One Virginia opossum (Didelphis virginiana) and 1 water opossum (Chironectes minimus) were submitted for postmortem examination from a zoo that housed 6 opossums, all of which died within several weeks. Red kangaroos (Macropus rufus) and red-necked wallabies (Macropus rufogriseus) were also present at the facility. Liver samples from both opossums were submitted for transmission electron microscopy and whole-genome sequencing. Microscopically, both opossums had multifocal necrosis in the liver and lung, with intranuclear inclusion bodies within hepatocytes and pneumocytes. Another significant finding in the Virginia opossum was sepsis, with isolation of Streptococcus didelphis from various organs. Ultrastructural analysis of formalin-fixed liver tissue identified herpesviral replication complexes in both opossums; negative-stain electron microscopy of unfixed liver tissue repeatedly yielded a negative result. The herpesvirus had >99% nucleotide identity with MaAHV2. These 2 cases indicate that both opossum species are susceptible to MaAHV2 infection, and the outbreak has implications for mixed-species facilities that house macropods.

Abstract 114: Inhibition Of Lecithin Cholesterol Acyl Transferase By A Novel Anti-apolipoprotein A1 Antibody Fragment

Apolipoprotein A1 (APOA1), the most abundant protein in high-density lipoproteins (HDL), is the primary activator of lecithin cholesterol acyl transferase (LCAT). LCAT’s cholesteryl esterification activity is key for maturing nascent HDL discs into the spherical forms commonly found in plasma. However, the molecular mechanism for APOA1 activation of LCAT is poorly understood. Using phage display selection against nanodiscs (lipid complexes stabilized by an APOA1 variant commonly used in transmembrane protein structural biology), we have identified, isolated, and expressed a novel Fab fragment (Fab16). Size exclusion chromatography elution shift experiments showed that Fab16 readily bound reconstituted HDL (rHDL) particles containing human purified plasma APOA1 with nearly quantitative binding at a 1:1 molar ratio of Fab16 to APOA1. Negative stain electron microscopy (NSEM), confirmed that 1-2 Fab16 bound to each rHDL particle in a specific pattern around the disc periphery. Additionally, Fab16 effectively bound both large and small spherical HDL particles isolated from human plasma. Chemical cross-linking / mass spectrometry experiments indicated that Fab16 binds an APOA1 epitope between residues 94 and 118 (i.e., within amphipathic helix 4). As our previous work has shown that APOA1 helix 4 is important for LCAT activation, we hypothesized that Fab16 would inhibit LCAT activity. Using a novel gas chromatography cholesterol esterification assay, we found that Fab16 applied at a 1.5:1 molar ratio to APOA1 inhibited LCAT cholesterol esterification activity by 56±7%. The symmetrical nature of both discoidal and spherical HDL particles has hampered the application of cryogenic electron microscopy (cryo EM) for understanding HDL structure. Fab16 may provide a valuable ‘landmark’ to assist in deriving a high-resolution structure of the complex. Work to optimize conditions for collecting high quality cryo EM datasets of discoidal and spherical HDL in the presence and absence of LCAT is ongoing. Deriving a high-resolution understanding of HDL structure and its interaction with remodeling factors such LCAT has been a long-standing problem but is crucial for deriving new strategies to manipulate HDL function for therapeutic benefit.

Alternative splicing controls teneurin-3 compact dimer formation for neuronal recognition

Neuronal network formation is facilitated by recognition between synaptic cell adhesion molecules at the cell surface. Alternative splicing of cell adhesion molecules provides additional specificity in forming neuronal connections. For the teneurin family of cell adhesion molecules, alternative splicing of the EGF-repeats and NHL domain controls synaptic protein-protein interactions. Here we present cryo-EM structures of the compact dimeric ectodomain of two teneurin-3 isoforms that harbour the splice insert in the EGF-repeats. This dimer is stabilised by an EGF8-ABD contact between subunits. Cryo-EM reconstructions of all four splice variants, together with SAXS and negative stain EM, reveal compacted dimers for each, with variant-specific dimeric arrangements. This results in specific trans-cellular interactions, as tested in cell clustering and stripe assays. The compact conformations provide a structural basis for teneurin homo- and heterophilic interactions. Altogether, our findings demonstrate how alternative splicing results in rearrangements of the dimeric subunits, influencing neuronal recognition and likely circuit wiring.

Milligram-scale assembly and NMR fingerprint of tau fibrils adopting the Alzheimer’s disease fold

In the Alzheimer’s disease (AD) brain, the microtubule-associated protein tau aggregates into paired helical filaments (PHFs) in which each protofilament has a C-shaped conformation. In vitro assembly of tau fibrils adopting this fold is highly valuable for both fundamental and applied studies of AD without requiring patient-brain extracted fibrils. To date, reported methods for forming AD-fold tau fibrils have been irreproducible and sensitive to subtle variations in fibrillization conditions. Here we describe a route to reproducibly assemble tau fibrils adopting the AD fold on the multi-milligram scale. We investigated the fibrilization conditions of two constructs, and found that a tau (297-407) construct that contains four AD phospho-mimetic glutamate mutations robustly formed the C-shaped conformation. Two- and three-dimensional correlation solid-state NMR spectra show a single predominant set of chemical shifts, indicating a single molecular conformation. Negative-stain electron microscopy and cryoelectron microscopy data confirm that the protofilament formed by 4E-tau (297-407) adopts the C-shaped conformation, which associates into paired, triple and quadruple helical filaments. In comparison, NMR spectra indicate that a previously reported construct, tau (297-391), forms a mixture of a four-layered dimer structure and the C-shaped structure, whose populations are highly sensitive to the environmental conditions. The determination of the NMR chemical shifts of the AD-fold tau opens the possibility for future studies of tau fibril conformations and ligand binding by NMR. The quantitative assembly of tau fibrils adopting the AD fold should facilitate the development of diagnostic and therapeutic compounds that target AD tau.

The HEAT repeat protein HPO-27 is a lysosome fission factor.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.