Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans, they cause extensive pathology following snakebite envenomation. In viper venom, some of the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain an SVMP cocktail and due to their cytotoxicity, SVMP characterisations have been hampered by the lack of purified enzymes. By incorporating their prodomain, which blocks the active SVMP site, we overcame their cytotoxicity and enabled recombinant production of zymogens from all three structurally variable SVMP classes (PI, PII, and PIII) using our baculovirus/insect cell expression system. Zymogens were auto-activated by incubation with Zn2+ ions, resulting in prodomain cleavage, PII disintegrin cleavage and PIII prodomain proteolysis. Auto-activated SVMPs were characterised using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our recombinant zymogen production protocol is generically applicable for the expression of SVMPs, unlocking biomedical use in haematology and discovery of novel snakebite therapeutics.

Virus-like particles (VLPs) are promising platforms for next-generation vaccines due to their ability to present antigens in highly ordered, repetitive geometries emulating pathogen-associated patterns to elicit potent immune responses. The ADDomer is a synthetic dodecahedral VLP scaffold derived from the penton base protein (PBP) of human adenovirus serotype 3 (Ad3). PBP tolerates insertion of multiple antigenic epitopes in flexible surface-exposed loops, and spontaneously self-assembles in vitro into ADDomer nanoparticles, but faces limitations including incomplete assembly and susceptibility to preexisting antihuman adenovirus immunity. Here, we report two complementary engineering strategies to enhance ADDomer robustness. First, we developed a Chimpanzee adenovirus Y25-based ADDomer (CHIMPSELS) to circumvent preexisting antihuman adenovirus immunity, and introduced a point mutation to restore a motif critical for dodecahedron integrity. Second, we introduced targeted intersubunit disulfide bonds to reinforce particle assembly. High-resolution electron cryo-microscopy confirmed the formation of intact dodecahedral particles, revealing that disulfide bonds stabilize distinct conformations of the PBP N-termini. Differential scanning fluorimetry and dynamic light scattering demonstrated thermal stability and elevated aggregation onset temperatures in the disulfide-stabilized ADDomers, providing a scalable assay for screening ADDomer-based VLP constructs for vaccine development. Incorporation of validated immunogenic epitopes, including a SARS-CoV-2 receptor-binding motif segment and the Chikungunya E2EP3 peptide, demonstrated structural integrity and epitope display by the modified scaffolds. Our results establish a versatile, thermostable VLP platform with reduced susceptibility to preexisting immunity, improved particle integrity, and capacity for modular epitope presentation. This work advances the ADDomer toward practical applications in vaccine development and highlights engineering strategies that can be broadly applied to enhance the performance of protein-based VLP vaccines.

FtsH, an essential AAA+ metalloprotease, maintains cellular homeostasis by degrading misfolded and membrane-associated proteins. Here, we report cryo-EM structures of the Escherichia coli FtsH periplasmic domain (FtsH-PD) revealing insights into its conformational flexibility. Initial 2D class averages suggested three distinct orientations, right-handed and left-handed maps of FtsH-PD, and a map with a different conformation. The 4.9 Å structure of FtsH-PD exhibits the conserved α+β fold, while the 7.3 Å map with the different conformation displays a 20º clockwise rotation of two alpha helices. These findings support a model where conformational changes are present not only in the FtsH cytosolic domain, but also in the periplasmic domain and potentially facilitate substrate translocation through a combination of mechanisms involving both the FtsH-PD and the HflKC complexed with FtsH, along with lipid-scramblase activity to assist in membrane protein extraction. This study points out novel perspectives on how conformational changes in the periplasmic domain contribute to FtsH substrate degradation mechanisms.

Chronic oxidative stress and lipid imbalance drive metabolic disorders such as obesity and non-alcoholic fatty liver disease, yet few therapies target the upstream redox imbalance in key tissues. Human carbonic anhydrase III (hCA III), a redox-associated enzyme enriched in liver and adipose tissue, has long remained pharmacologically elusive due to its low catalytic activity and lack of modulators. Here, we identify fragment-like nicotinic acid derivatives as non-sulfonamide hCA III modulators and evaluate their associated cellular effects. Using an esterase activity assay, we screened 25 analogues and identified two fragment-like hits, compound 17 (2-thioethyl) and compound 22 (6-morpholino), with IC50 values of 487 and 361 µM, respectively. Orthogonal thermal shift analysis supported compound-protein interaction, and selected hits were subsequently evaluated in HepG2 cells. Both compounds were associated with reduced CA3 mRNA expression after treatment at 1 µM, while their cellular phenotypes diverged, with compound 22 increasing ROS under oxidative stress conditions and compound 17 affecting mitochondrial membrane potential. Taken together, these findings identify tractable nicotinic acid-derived fragment hits and associated cellular phenotypes that warrant further mechanistic investigation. These fragment-like hits provide a practical starting point for studying the redox-linked biology of hCA III.

Current lab-scale purification methods for ADDomer are labour-intensive, time-consuming and poorly scalable. The intracellular nature of ADDomer production further complicates downstream processing, requiring robust, scalable solutions for cell lysis and clarification. In the present work we focus on developing a scalable, GMP-compliant process for ADDomer purification. The workflow combines tangential microfiltration (TMF) using a 0.4 μm hollow fiber for cell retention, chemical lysis with 0.06

pH critically influences SARS-CoV-2 infectivity and stability by, for example, triggering pH-dependent conformational changes in the spike glycoprotein that can facilitate membrane fusion and viral entry into host cells. Using the emerging dynamical nonequilibrium molecular dynamics (D-NEMD) simulations approach, we investigated how biologically relevant pH shifts affect the functional dynamics of the fully glycosylated spike from the ancestral, Delta, and Omicron BA.1 variants of concern. For this, over 1100 nonequilibrium simulations were conducted to capture the pH-induced structural and dynamic changes that occur following transitions from physiological to acidic and alkaline conditions, with the former mimicking the low pH environment within endosomes, and the latter the high pH conditions accessible to nascent exhaled aerosols. D-NEMD reveals that pH changes trigger distinct, variant-specific conformational responses in key regions of the spike, including the receptor binding domain (RBD), fusion peptide proximal region (FPPR), and C-terminal domain (CTD). The ancestral spike shows broad pH sensitivity, characterised by directional motions and region-specific structural rearrangements that depend on the pH conditions. The spike of the Delta variant displays increased reactivity to alkaline pH, potentially explaining its reduced stability in alkaline aerosols. The spike of Omicron BA.1, in contrast, responds strongly to acidic conditions with spontaneous RBD opening and pronounced structural rearrangements in the FPPR and CTD. This behaviour aligns with this variant’s preference for endosomal entry and reduced reliance on TMPRSS2-mediated fusion. The Omicron BA.1 spike also shows increased resilience to alkaline pH, suggesting greater environmental stability. Our findings further emphasise the key role of glycans in spike activation, with glycan N234 stabilising the RBD “up” conformation during pH-induced transitions in Omicron under acidic conditions. These insights, together, highlight pH as a potential evolutionary pressure for SARS-CoV-2 and underscore the importance of glycosylation and environmental pH variability in shaping the behaviour of viral fusion proteins. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BB/X009831/1, BB/W003449/1, BB/Z516533/1, BB/W00884X/1 European Research Council, Grant agreement No. 101021207; project information: PREDACTED NSF RAPID, MCB-2032054

Following our recent identification of a fatty acid binding site in the SARS-CoV-2 spike protein (Toelzer et al., Science eabd3255 (2020)), we investigate the binding of linoleate and other potential ligands at this site using molecular dynamics simulations. The results support the hypothesis that linoleate stabilises the locked form of the spike, in which its interaction interface for the ACE2 receptor is occluded. The simulations indicate weaker binding of linoleate to the partially open conformation. Simulations of dexamethasone bound at this site indicate that it binds similarly to linoleate, and thus may also stabilize a locked spike conformation. In contrast, simulations suggest that cholesterol bound at this site may destabilize the locked conformation, and in the open conformation, may preferentially bind at an alternative site in the hinge region between the receptor binding domain and the domain below, which could have functional relevance. We also use molecular docking to identify potential ligands that may bind at the fatty acid binding site, using the Bristol University Docking Engine (BUDE). BUDE docking successfully reproduces the linoleate complex and also supports binding of dexamethasone at the spike fatty acid site. Virtual screening of a library of approved drugs identifies vitamins D, K and A, as well as retinoid ligands with experimentally demonstrated activity against SARS-CoV-2 replication in vitro, as also potentially able to bind at this site. Our data suggest that the fatty acid binding site of the SARS-CoV-2 spike protein may bind a diverse array of candidate ligands. Targeting this site with small molecules, including dietary components such as vitamins, which may stabilise its locked conformation and represents a potential avenue for novel therapeutics or prophylaxis for COVID-19.

Snakebite envenoming constitutes a significant global health issue, particularly in Africa, where venomous species such as Echis vipers and Dendroaspis mambas pose substantial risks to human health. This study employs a standardized venomics workflow to comprehensively characterize and comparatively quantify the venom composition of nine medically relevant snake species chosen from among the deadliest in Africa. Utilizing shotgun venom proteomics and venom gland transcriptomics, we report detailed profiles of venom complexity, highlighting the relative abundance of dominant toxin families such as three-finger toxins and Kunitz-type proteins in Dendroaspis, and metalloproteinases and phospholipases A2 in Echis. We delineate here the relative abundance and structural diversity of venom components. Key to our proteomic approach is the implementation of Multi-Enzymatic Limited Digestion (MELD), which improved protein sequence coverage and enabled the identification of rare toxin families such as hyaluronidases and renin-like proteases, by multiplying the overlap of generated peptides and enhancing the characterization of both toxin and non-toxin components within the venoms. The culmination of these efforts resulted in the construction of a detailed toxin database, providing insights into the biological roles and potential therapeutic targets of venom proteins and peptides. The findings here compellingly validate the MELD technique, reinforcing its reproducibility as a valuable characterization approach applied to venomics. This research significantly advances our understanding of venom complexity in African snake species, including representatives of both Viperidae and Elapidae families. By elucidating venom composition and toxin profiles, our study paves the way for the development of targeted therapies aimed at mitigating the morbidity and mortality associated with snakebite envenoming globally.

The spike protein is essential to the SARS-CoV-2 virus life cycle, facilitating virus entry and mediating viral-host membrane fusion. The spike contains a fatty acid (FA) binding site between every two neighbouring receptor-binding domains. This site is coupled to key regions in the protein, but the impact of glycans on these allosteric effects has not been investigated. Using dynamical nonequilibrium molecular dynamics (D-NEMD) simulations, we explore the allosteric effects of the FA site in the fully glycosylated spike of the SARS-CoV-2 ancestral variant. Our results identify the allosteric networks connecting the FA site to functionally important regions in the protein, including the receptor-binding motif, an antigenic supersite in the N-terminal domain, the fusion peptide region, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected link between different allosteric sites. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spike revealed that glycans do not qualitatively change the internal allosteric pathways but can facilitate the transmission of the structural changes within and between subunits.

BACKGROUND:Immunological memory to vaccination and viral infection involves the coordinated action of B and T cells; thus, integrated analysis of these 2 components is critical for understanding their respective contributions to protection against breakthrough infections (BIs) after vaccination. METHODS:We investigated cellular and humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and/or vaccination in 300 adult participants from the Avon Longitudinal Study of Parents and Children (ALSPAC). Participants were grouped by those with (cases) and without (controls) a history of SARS-CoV-2 infection. To provide a quantitative correlate for protection against BI in the 8-month period after the study, Youden index thresholds were calculated for all immune measures analyzed. RESULTS:The magnitude of antibody and T-cell responses following the second vaccine dose was associated with protection against BI in participants with a history of SARS-CoV-2 infection (cases), but not in infection-naive controls. Over 8 months of follow-up, 2 threshold combinations provided the best performance for protection against BI in cases: (i) anti-spike immunoglobulin G (IgG) (≥666.4 binding antibody units [BAU]/mL) combined with anti-nucleocapsid pan-immunoglobulin (pan-Ig) (≥0.1332 BAU/mL) and (ii) spike 1-specific T cells (≥195.6 spot-forming units/106 peripheral blood mononuclear cells) combined with anti-N pan-Ig (≥0.1332 BAU/mL). Both combinations offered 100% specificity for detecting cases without BI, with sensitivities of 83.3% and 72.2%, respectively. CONCLUSIONS:Collectively, these results suggest that hybrid B- and T-cell immunity offers superior protection from BI after coronavirus disease 2019 (COVID-19) vaccination, and this finding has implications for designing next-generation COVID-19 vaccines that are capable of eliciting immunity to a broader repertoire of SARS-CoV-2 proteins.

Transcription initiation involves the coordination of multiple events, starting with activators binding specific DNA target sequences, which recruit transcription coactivators to open chromatin and enable binding of general transcription factors and RNA polymerase II to promoters. Two key human transcriptional coactivator complexes, ATAC (ADA-two-A-containing) and SAGA (Spt-Ada-Gcn5 acetyltransferase), containing histone acetyltransferase (HAT) activity, target genomic loci to increase promoter accessibility. To better understand the function of ATAC and SAGA HAT complexes, we used in vitro biochemical and biophysical assays to characterize human ATAC and SAGA HAT module interactions with nucleosomes and how a transcription factor (TF) coordinates these interactions. We found that ATAC and SAGA HAT modules bind nucleosomes with high affinity, independent of their HAT activity and the tested TF. ATAC and SAGA HAT modules directly interact with the VP16 activator domain and this domain enhances acetylation activity of both HAT modules. Surprisingly, ATAC and SAGA HAT modules increase TF binding to its DNA target site within the nucleosome by an order of magnitude independent of histone acetylation. Altogether, our results reveal synergistic coordination between HAT modules and a TF, where ATAC and SAGA HAT modules (i) acetylate histones to open chromatin and (ii) facilitate TF targeting within nucleosomes independently of their acetylation activity.

Snake venoms contain variable mixtures of toxins that evolved to incapacitate prey but cause extensive pathology in snakebite patients. In viper venom, the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). SVMP research has been hampered by the lack of an efficient generic recombinant production protocol. Using baculovirus/insect cell expression, we produced and functionally validated enzymes from all three structurally variable SVMP classes (PI, PII and PIII). Incorporating the native N-terminal prodomain, which blocks the active site via a cysteine-switch motif, overcame the cytotoxicity of SVMPs. Incubation with Zn2+ activated the SVMP zymogens, resulting in proteolysis of the PIII prodomain. Functional validation of the recombinant SVMPs was performed using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our study provides a potent generic platform for the expression of SVMPs of value, for bioprospecting and discovery of novel snakebite therapeutics. ### Competing Interest Statement The authors have declared no competing interest. Source data are provided with this paper. European Council, 899670, 101149867 Wellcome Trust, https://ror.org/029chgv08, 221708/Z/20/Z Biotechnology and Biological Sciences Research Council, BB/Y007581/1 Engineering and Physical Sciences Research Council, EP/Z002613/1

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway involved in translational control and gene expression regulation. Core NMD factors up-frameshift proteins UPF1, UPF2, and UPF3B are conserved from yeast to humans and essential to target mRNAs with a premature stop codon for decay. UPF2 binding to UPF1 activates UPF1's ATPase and helicase activities, and UPF2 binding to UPF3B is important for its association with the exon junction complex and efficient NMD. However, UPF2's association with RNA remains largely uncharacterized. Here, we analyze nucleic acid binding, identifying the first and third MIF4G domains of UPF2 as main RNA-/DNA-binding modules. We find that UPF2's MIF4G domain-3 has RNA annealing activity, while full-length UPF2 unfolds our reporter hairpin RNA structure. We show that UPF2 preferentially binds and stabilizes single-stranded RNA (ss-RNA) in a sequence-independent manner. Concomitant to ss-RNA binding, UPF2 undergoes a distinct conformational change in its otherwise highly dynamic structure. UPF2's RNA binding and unfolding activity may support UPF1's helicase and messenger ribonucleoprotein remodeling activity and, in combination with UPF3B, stabilize UPF1's association with nonsense mRNA.

Horseradish peroxidase (HRP), isolated from horseradish roots, is heavily glycosylated, making it difficult to crystallize. In this work, we produced recombinant HRP in E. coli and obtained an X-ray structure of the ferric enzyme at 1.63 Å resolution. The structure shows that the recombinant HRP contains four disulphide bonds and two calcium ions, which are highly conserved in class III peroxidase enzymes. The heme active site contains histidine residues at the proximal (His 170) and distal (His 42) positions, and an active site arginine (Arg 38). Surprisingly, an ethylene glycol molecule was identified in the active site, forming hydrogen bonds with His 42 and Arg 38 at the δ-heme edge. The high yields obtained from the recombinant expression system, and the successful crystallization of the enzyme pave the way for new structural studies in the future.

Coenzyme Q10 (Q10) plays a critical role in cellular energy conversion within the mitochondrial respiratory chain and offers protective effects against oxidative and metabolic stress. In this study, we investigated the impact of Q10 on the spatio-temporal patterns of cellular energetics in keratinocyte-derived HaCaT cells, utilizing the genetically-encoded FRET sensor AMPfret. Engineered from the AMP-activated protein kinase (AMPK), this sensor leverages endogenous affinities of the kinase that evolved to detect energy stress, specifically decreases in ATP/ADP and ATP/AMP ratios that pose a threat to cell survival. We successfully established HaCaT cells stably expressing AMPfret, validated their functionality by inducing energy stress with 2-deoxy-D-glucose, and demonstrated that Q10, together with high glucose conditions in culture, can enhance cellular energetics compared to low glucose controls. We then employed AMPfret to analyze the spatio-temporal response of HaCaT keratinocytes to Luperox (tert-butyl peroxide), a potent organic prooxidant, in the presence of varying intracellular levels of Q10. Preloading cells with Q10 was protective, slowing the speed and reducing the extend of the energy stress response. In contrast, preincubation with Simvastatin, an inhibitor of the mevalonate Q10 biosynthesis pathway, depleted cellular Q10 levels, accelerated the onset of energy stress, and led to early cell death as compared to controls. Under all conditions, AMPfret revealed cell-to-cell heterogeneity in energy stress at baseline and in the response to Luperox. Overall, tracking changes in energy state in time and at single-cell level allows further insights into the beneficial role of Q10 in enhancing cellular bioenergetics in skin cells, and a potential role of AMPK in mediating responses to altered Q10 levels.