Biology subjects

Struwe, W. B.

Publications and source records attributed to Struwe, W. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Human Choroid Plexus Organoids Progress to Postnatal-like Maturation and Reveal a Closing Window of Developmental Plasticity

The choroid plexus (ChP) produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier, yet the developmental programme driving its maturation in humans remains poorly understood. Using a 15-timepoint single-cell RNA sequencing atlas of human ChP organoids benchmarked against human tissue, we resolve an ordered differentiation process comprising cell cycle exit, ciliogenesis and ion transporter expression. Maturation culminates in a transcriptional state with features of human adult ChP, establishing the first in vitro ChP model to reach postnatal-like milestones. Centrosome proteomics identified previously uncharacterised, stage-specific regulators of ciliogenesis. To probe developmental plasticity, we perturbed ChP organoids with valproic acid (VPA), a clinically used antiepileptic and established teratogen. Early VPA-mediated disruption of multiciliogenesis and secretion recovered upon drug withdrawal, whereas late treatment washout resulted in persistent impairment of mature transporters. Together, these findings resolve the developmental programme by which the ChP reaches functionally mature identity and identify a stage-dependent plasticity window during its maturation.

developmental biology↗

bioRxiv2026

Mechanism of spike opening in a mammalian gammacoronavirus

Coronavirus spike proteins mediate receptor binding and fusion and are key determinants of viral host range. To bind protein receptors, some CoV spikes undergo conformational changes termed opening, which expose the receptor-binding surface. Yet, the prerequisites and mechanism of spike opening remain poorly understood, especially outside of beta-CoVs, and many CoV spikes have been observed only in the closed conformation. The receptor specificities of gamma-CoVs, for which birds are natural hosts, await in-depth characterisation and their spikes have not been observed to open. Here, we report structures of the spike from an unusual mammalian gamma-CoV isolated from a beluga whale (BwCoV), in closed and open conformations, the latter with two S1B domains erect. BwCoV spike opening is associated with changes throughout S1 that resemble those observed in beta-CoV spikes and similarly propagate into destabilisation of the fusogenic S2 core. Furthermore, the BwCoV-S structure contains an additional density likely corresponding to a fatty acid, consistent with lipid binding stabilising the closed conformation through a pocket distinct from that identified for beta-CoVs. These structural insights reveal conserved principles regulating spike dynamics across coronavirus genera and support lipid binding as a potential modulatory mechanism of spike opening.

biochemistry↗

bioRxiv2026

An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin

O-glycosylation is among the most abundant and structurally diverse post-translational modifications in eukaryotes, yet its heterogeneity renders O-glycoproteins exceptionally difficult to characterize. To overcome these challenges, we have developed an integrated mass spectrometry (MS) strategy to define the proteoform landscape of O-glycoproteins and applied it to human osteopontin (OPN). OPN is a disease-associated extracellular matrix protein subject to extensive modification. By combining native MS with serial exoglycosidase digestions, we directly resolved truncation, phosphorylation, sulfation, and O-glycosylation of OPN. Matched glycoproteomic analyses, using tailored (glyco)protease combinations, allowed us to quantify glycan heterogeneity inaccessible to conventional trypsin-based approaches or protein-centric methods. We integrated these datasets using forward compositional simulations to infer the intact OPN proteoform distribution and benchmarked the resulting models against an experimental intact-mass distribution obtained by proton-transfer charge-reduction MS. This comparison revealed that bottom-up O-glycoproteomics systematically underestimates the true extent of glycan sialylation, whereas assuming (near-)complete sialylation accurately reproduced the experimental intact OPN mass distribution. Together, these results provide a comprehensive, quantitative view of OPN compositional diversity and demonstrate how intact-protein and peptide-level measurements can be reconciled to resolve highly heterogeneous glycoform populations. The workflow establishes a broadly applicable framework for characterizing extensively O-glycosylated and multiply modified proteins.

biophysics↗

bioRxiv2026

Allosteric remodelling of the Ebola virus glycoprotein underlies differences in entry mechanisms across species

During Ebolavirus transit through the endo-lysosomal pathway, the glycoprotein GP undergoes a series of conformational rearrangements upon cathepsin cleavage and NPC1 receptor binding, culminating in host-virus membrane fusion. While these rearrangements underpin viral entry, their molecular mechanisms remain poorly understood. Here, we combine hydrogen/deuterium-exchange mass spectrometry and mass photometry to resolve structural dynamic and energetic transitions of GP of two major species, Zaire (EBOV) and Sudan (SUDV). We describe the allosteric axes that govern reorganization of the cleavage sites, opening of the receptor binding cavity and priming for fusion upon NPC1 engagement. We show that GP cathepsin cleavage and receptor binding exhibit different mechanisms and kinetics across species, and are allosterically coupled in a species-specific manner. We reveal that fusion priming is optimized via distinct routes in EBOV and SUDV GP. These data suggest that GP structural dynamics and allosteric remodelling underlie differences in entry mechanisms across Ebolavirus species.

biochemistry↗

bioRxiv2026

LemonCatcher Acidic Pull-Down Enables Selective In-Cell Hydrogen-Deuterium Exchange Mass Spectrometry

Proteins are dynamic molecules which sensitively adapt according to their environment. Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) provides unique insights into protein conformational processes. However, existing methodology cannot selectively enrich proteins post-labeling because D-to-H back exchange must be minimized by rapid processing at pH 2.3-3.0 and 0 {degrees}C, where affinity purification fails. Here, we create LemonCatcher, a protein superglue that spontaneously forms an amide bond to the LemonTag peptide under these harsh acidic and cold quench conditions, even at -20 {degrees}C. Engineering of a bead-coupled LemonCatcher purification system introduces fast and selective quench-capture HDX-MS (SelQueX) on LemonTagged fusion proteins. We demonstrate targeted measurement of protein dynamics in living bacterial cells, revealing ligand-induced conformational changes in maltose-binding protein. Moreover, probing a stalled membrane protein nascent-chain supports a role for the ribosome in maintaining partially unfolded folding intermediates. Thus, SelQueX makes possible selective characterization of protein structural dynamics within the complex cellular milieu.

biochemistry↗

bioRxiv2026

A proteomic feasibility study connecting metabolic and synaptic pathway alterations in serum and extracellular vesicles to characterize treatment-resistant depression

Treatment-resistant depression (TRD) remains a major clinical challenge, yet the biological processes distinguishing TRD from non-treatment-resistant depression (nTRD) are incompletely defined. While circulating serum proteomes reflect broad systemic alterations associated with depression, extracellular vesicles (EVs) could provide a more selective representation of intercellular signaling relevant to treatment resistance. Here, we carried out a pilot study to evaluate the extent that parallel proteomic profiling of serum and serum-derived EVs could distinguish healthy controls (CON), nTRD, and TRD individuals. In this exploratory and hypothesis-generating study, serum proteomes exhibited robust global differences between depression groups and controls, largely reflecting shared systemic biology across nTRD and TRD. In contrast, EV proteomes showed limited global separation but revealed subtype-associated pathway differences. Relative to controls, nTRD EVs were enriched for immune and inflammatory pathways. By contrast, TRD EVs were characterized by enrichment of mitochondrial metabolism, oxidative phosphorylation, translational initiation, and MYC-regulated pathways, together with depletion of synaptic signalling, membrane trafficking, and cytoskeletal pathways. Comparative analysis of pathways significant in both contrasts revealed that these bioenergetic and translational signatures were selectively amplified in TRD relative to nTRD. Our exploratory analyses identified that the circulating EV cargo may reflect a treatment-resistance-specific reorganization of biological pathways not apparent in bulk serum proteomics. This study highlights parallel serum and EV proteomics as a complementary approach for molecular stratification in antidepressant resistance.

neuroscience↗

bioRxiv2026

Selective miniprotein inhibitors of Aurora-A kinase designed using interaction-motif scaffolding

Targeting kinase ATP-binding sites has produced many successful therapeutics, but selectivity remains a major challenge. We hypothesised that substrate-recognition surfaces could provide an alternative route to selective kinase inhibition. Here, we designed and tested structure-guided miniprotein inhibitors of Aurora-A using N-Myc as a template, a natural weak binder of the P+1 pocket. The most potent designs bind Aurora-A with single-digit nanomolar affinity, more than a thousand fold higher than the starting template, and selectively inhibit Aurora-A over Aurora-B in kinase assays and mitotic cells. Despite diverse architectures, successful binders targeting the substrate-recognition surface converged on a common strategy, coupling extensive engagement of the G helix with activation loop stabilisation. Together these results show that selective kinase inhibition can be achieved by exploiting structurally well-defined active-state conformations rather than kinase-specific inactive states. Our work establishes a framework for converting weak substrate-recognition surface interactors into potent, selective kinase inhibitors through structure-guided protein design.

molecular biology↗

bioRxiv2026

Molecular dissection of protein complexes isolated from sections of human brain

Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop nanobody-based immunoprecipitation coupled with native mass spectrometry to interrogate endogenous protein assemblies from post-mortem mouse and human brain sections. We exemplify our approach by characterizing the synaptic proteins VGluT1 and mGluR2. From a single mouse brain, we discover mGluR2/3 heterodimers, alongside mGluR2 homodimers. Considering regions of human brain heterodimeric mGluR2/3 is highly abundant in the OFC and sgACC ([~]70% and 50%, respectively) and forms regional-specific interactions with additional synaptic proteins. In a modest cohort of biobanked human tissue, associated with depression and suicide, we find increased mGluR2/3 in the OFC. Consistent with this, mice exhibit similar associations between heterodimer levels and stress-susceptibility. Overall, our approach provides a direct means for establishing molecular-behavioural links at the level of receptor organization in brain.

biochemistry↗

bioRxiv2026

Continuous mass photometry by single molecule trapping

The dynamic choreography of biomolecular interactions underpins the processes of life, but its direct observation remains challenging. Here, we introduce confined diffusion mass photometry, enabling hour-long, mass-resolved observation of individual biomolecules, their complexes and interactions with up to sub-kDa mass precision and ms temporal resolution. Our approach represents a quantitative time-resolved single-molecule measurement modality for studying complex biomolecular mechanisms in action.

biophysics↗

bioRxiv2026

Iterative Bump-and-hole engineering creates a bioorthogonal reporter for N-acetylglucosaminyltransferase I

Asparagine-linked protein glycosylation is among the most frequent modifications of proteins trafficking through the secretory pathway. These glycans are manufactured in an assembly line process to a common precursor that is then subject to individual modifications with different levels of complexity. An important biosynthetic modulator is the incorporation of N-acetylglucosamine (GlcNAc) at distinct positions in N-linked glycan biosynthesis, commencing with the activity of the glycosyltransferase MGAT1. While mapping of N-glycans to their corresponding protein attachment sites is generally possible, not much is known about the glycoprotein substrate choice for MGAT1 and related transferases. Analogs of GlcNAc with small bioorthogonal tags can be incorporated into N-glycans. However, due to the promiscuity of some GlcNAc transferases, incorporation is of little specificity towards individual positions. Here, we report an iterative bump-and-hole approach in the design of a bioorthogonal precision tool for the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 towards the nucleotide-sugar UDP-GlcNAc while retaining activity towards bumped, azide-modified analogs. Kinetic and computational analyses using a neural network approach informed the synthesis of a tailored UDP-GlcNAc analog with preferential acceptance by the engineered enzyme. Following substrate biosynthesis, the strategy allowed selective incorporation of a chemical tag on MGAT1 substrate proteins in living mammalian cells with little background incorporation by other GlcNAc transferases. Our work expands the toolbox for glycan-based reporter compounds.

biochemistry↗

bioRxiv2025

An Evolutionary Distinct Nipah G Glycosylation Site Provides Stability for Receptor Engagement

Nipah virus is a deadly paramyxovirus with 40-75% mortality and >750 cases since 1998. Currently there are no clinically approved vaccines or therapeutics to target infection. Nipah is an enveloped virus with two surface glycoproteins, the trimeric fusion (F) and tetrameric attachment glycoprotein (G). G is responsible for cellular attachment via binding to ephrin B2/B3. Glycosylation of Nipah G and its effects on receptor engagement has not previously been studied but is important as glycosylation impacts immunogenicity, receptor binding and structural conformations for other enveloped virus glycoproteins. Our phylogenetic and mass spectrometry analysis of site-specific N-glycans of the Nipah G Malaysia strain revealed how N-glycosylation has evolved since the appearance of the virus in 1998. We discovered that the N481 N-glycosite is not conserved and although the glycan does not directly contribute to receptor binding, the threonine/serine in the glycosylation sequon is critical for maintaining long-range stability of individual G subunits that facilitates ephrin B2 binding affinity. Together, these data reveal plasticity of N-glycosylation sites across Nipah species and the presence of hydrogen bonding networks that contribute to G stability and host engagement, which is valuable information for understanding virus attachment/entry mechanisms as well as the rationale design of structure-based vaccines. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/646399v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@15960a8org.highwire.dtl.DTLVardef@d8cacdorg.highwire.dtl.DTLVardef@ea6eb4org.highwire.dtl.DTLVardef@10a2c84_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Structural and Functional Glycosylation of the Abdala COVID-19 Vaccine

Abdala is a COVID-19 vaccine produced in Pichia pastoris and is based on the receptor-binding domain (RBD) of the SARS-CoV-2 spike. Abdala is currently approved for use in multiple countries with clinical trials confirming its safety and efficacy in preventing severe illness and death. Although P. pastoris is used as an expression system for protein-based vaccines, yeast glycosylation remains largely uncharacterised across immunogens. Here, we characterise N-glycan structures and their site of attachment on Abdala and show how yeast-specific glycosylation decreases binding to the ACE2 receptor and a receptor-binding motif (RBM) targeting antibody compared to the equivalent mammalian-derived RBD. Reduced receptor and antibody binding is attributed to changes in conformational dynamics resulting from N-glycosylation. These data highlight the critical importance of glycosylation in vaccine design and demonstrate how individual glycans can influence host interactions and immune recognition via protein structural dynamics.

biochemistry↗

bioRxiv2024

Open and closed conformations of a sub-80 kDa Chagas vaccine candidate defined by a cryo-EM led integrative approach

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a significant global public health concern. Despite its profound health impact in both endemic and non-endemic areas, no vaccine is available, and the existing therapies are outdated, producing severe side effects. The 80kDa prolyl oligopeptidase of Trypanosoma cruzi (TcPOP) has been recently identified as a leading candidate for Chagas vaccine development. We report the first three-dimensional structure of TcPOP in open and closed conformation, at a global resolution of 3.8 and 3.6 [A] respectively, determined using single-particle cryo-electron microscopy. Multiple conformations were observed and further characterized using plasmonic optical tweezers and hydrogen-deuterium exchange mass spectrometry. To assess the immunogenic potential of TcPOP, we immunized mice and evaluated both polyclonal and monoclonal responses against the TcPOP antigen and its homologues. The results revealed invasion blocking properties of anti-TcPOP polyclonal response via parasite lysis and its cross-reactivity versus closely-related POPs but not with human homologues. We were also able to produce and characterise three monoclonal antibodies, one of which showed neutralising properties and inhibition of parasite invasion via non-lytic mechanism. Collectively, our findings provide critical structural and functional insights necessary to understand the immunogenicity of TcPOP for future Chagas vaccine development and diagnostic applications.

biophysics↗

bioRxiv2023

Haptenic adducts of beta-lactam antibiotics elicit antibody responses with narrow clonality and specificity

Many classes of small-molecule drugs form protein adducts in vivo, which may elicit antibodies via a classical hapten-carrier-type response, with implications for both allergy and drug sequestration. Although {beta}-lactam antibiotics are a drug class long associated with these phenomena, the molecular determinants of drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, we interrogated factors influencing penicilloyl adduct formation and immunogenicity, and used penicillin G (PenG) to probe the B and T cell determinants of drug-specific IgG responses in mice. We identify through deep clonotyping a dominant murine penicilloyl-specific clonal antibody class encompassing phylogenetically related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Through protein NMR and x-ray structural analysis, we determined that adduct specific antibody clones--the MIL series--predominantly recognise the variable side-chain moiety (which for PenG is phenylacetamide) via a hydrophobic pocket, while secondary H-bond contacts with both thiazolidine and the adducted lysine residue is made. As a result, the cross-reactivity against other {beta}-lactam antibiotics is limited. These data demonstrate the relationship between the chemistry of protein-reactive drugs such as penicilloyls, and how their predisposition to generating B cell responses can inform the functional implications at the clonal level. Highlights- PenG readily forms immunogenic adducts on lysine sidechains of diverse self- and non-self proteins including complete serum under physiological conditions. - PenG-protein adduction in vitro or in vivo is sufficient to elicit penicillin-specific IgG responses. - Murine B cell clonotypic responses are characterised by near-uniform antibody binding modes of similar immunogenetic origin. - The dominant murine PenG-specific clonotype is dominated by benzene ring recognition and correlates with serological cross-reactivity profiles.

immunology↗

bioRxiv2023

Cooperativity and induced oligomerisation control the interaction of SARS- CoV-2 with its cellular receptor and patient-derived antibodies

Viral entry is mediated by oligomeric proteins on the virus and cell surfaces. The association is therefore open to multivalent interactions between these proteins, yet such recognition is typically rationalised as affinity between monomeric equivalents. As a result, assessment of the thermodynamic mechanisms that control viral entry has been limited. Here, we use mass photometry to overcome the analytical challenges consequent to multivalency. Examining the interaction between the spike protein of SARS-CoV-2 and the ACE2 receptor, we find that ACE2 induces oligomerisation of spike in a variant-dependent fashion. We also demonstrate that patient-derived antibodies use induced-oligomerisation as a primary inhibition mechanism or to enhance the effects of receptor-site blocking. Our results reveal that naive affinity measurements are poor predictors of potency, and introduce a novel antibody-based inhibition mechanism for oligomeric targets. One-Sentence SummaryMultivalent interactions between viral proteins, cell-surface receptors, and anti-viral antibodies regulate infection and inhibition.

biophysics↗

bioRxiv2020

Analysis of SARS-CoV-2 spike glycosylation reveals shedding of a vaccine candidate

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of Nov 15, 2020 has claimed 1,319,946 lives worldwide. Vaccine development focuses on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralisation. Immunogen integrity is therefore important for glycoprotein-based vaccine candidates. Here we show how site-specific glycosylation differs between virus-derived spikes and spike proteins derived from a viral vectored SARS-CoV-2 vaccine candidate. We show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, which may have implications for the resulting immune response and future vaccine design.

biochemistry↗

bioRxiv2020

Hyper-Truncated N-Acetyl-β-D-Glucosamine Decorations Augment the Activity and Inhibition Potential of Neutrophil Granule Myeloperoxidase

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remain elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a previously unreported low-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility, and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, structural modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity. SignificanceMyeloperoxidase (MPO) is an important microbicidal glycoprotein critical for fighting pathogens. We report, for the first time, the intriguingly complex relationship between glycobiology and MPO immune function by demonstrating that uncommon and strategically positioned hyper-truncated glycans both elevate the activity and the inhibition potential of this pathogen-combating enzyme. We have used a multifaceted approach employing integrated biomolecular analytics to generate new insights into the sugar code of MPO. The findings described in this study improve our understanding of key innate immune processes and may guide future glycoengineering efforts aiming to generate therapeutically relevant recombinant MPO products with tuneable activity and inhibition potential tailored to biomedical applications involving persisting and severe pathogen infections.

biochemistry↗

bioRxiv2020

Correlating glycoforms of DC-SIGN with stability using a combination of enzymatic digestion and ion mobility MS

The immune scavenger protein DC-SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC-SIGN have not been reported. Here, we develop and apply a mass spectrometry-based approach to both uncover and characterise the effects of O-glycans on the stability of DC-SIGN. We first quantify the Core 1 & 2 O-glycan structures on the carbohydrate recognition and extracellular domains of the protein via sequential exoglycosidase sequencing. We then use ion mobility mass spectrometry to show how specific O-glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas-phase stability of the glycoprotein isoforms of DC-SIGN. We find that rather than the mass or length of glycoprotein modifications, the stability of DC-SIGN is better correlated with the number of glycosylation sites. Collectively, our results exemplify a combined multi-dimensional MS approach, proficient in evaluating protein stability in response to both glycoprotein macro- and micro-heterogeneity and adding structural detail to the infection enhancer DC-SIGN.

biophysics↗