Dengue virus (DENV) continues to impose a global health burden, and virus-like particles (VLPs) are promising vaccine candidates, owing to their ability to elicit broadly neutralizing antibodies. However, the lack of nanoscale structural insights into the VLP maturation process has limited rational engineering. Here, we report the cryo-electron microscopy (cryo-EM) structure of immature DENV serotype 2 VLPs, revealing prM-E spikes arrayed on a T = 1 shell, consistent with mature DENV-2 VLPs and distinct from the virion, which exhibits a T = 3 icosahedral lattice. To connect the experimentally determined immature and mature endpoint structures, we employed a multiscale molecular dynamics (MD) framework to assess sterically feasible transition pathways. The simulations support the steric feasibility of a sliding-rotating rearrangement in which trimeric prM-E spikes reorganize into flat E dimers (E-E) without clashes. In virions, trimers reorganize into extended rafts of three parallel E dimers, whereas in VLPs, which lack the long-range symmetry and geometric constraints of the T = 3 lattice, maturation proceeds via a less extensive rearrangement in which neighboring monomers form small triangular clusters of three dimers. In addition, the simulations reveal pronounced lipid core mobility during maturation, including transient and spatially localized lipid protrusion events that preferentially occur near regions undergoing protein rearrangement, consistent with a potential role for dynamic membrane remodeling in accommodating maturation-associated structural changes. We also established a stable Chinese hamster ovary (CHO-K1) producer cell line, enabling efficient production of immature DENV serotype 2 VLPs. Together, this work defines a structure-dynamics framework that links steric feasibility, membrane composition, and particle stability and outlines process-relevant, testable hypotheses to inform future engineering of dengue VLPs that may ultimately guide vaccine design.

Coarse-grained (CG) molecular dynamics enables the simulation of large biomolecular systems over extended timescales, but recovering atomistic detail from CG representations remains a significant challenge. Here, we present BackMapNet, a generalised and computationally e!cient framework for protein backmapping. The method adopts a staged reconstruction strategy for backbone and side-chain prediction. To enhance structural accuracy and physical realism, BackMapNet integrates geometric priors and Ramachandran-guided refinement to constrain backbone torsion angles. The model was trained on molecular dynamics trajectories from twelve structurally diverse proteins. Without retraining, BackMapNet was evaluated on previously unseen proteins. Across these systems, the model consistently achieved sub-Å accuracy, with overall protein RMSDs remaining below 0.85 Å for all test cases. Ramachandran-based refinement reduced torsional outliers while preserving coordinate fidelity. Benchmarking against established methods demonstrates that BackMapNet achieves competitive performance. These results indicate that accurate and transferable protein backmapping can be achieved with lightweight, non-equivariant architectures, provided that appropriate geometric priors and staged reconstruction strategies are employed.

Non-enveloped viruses exhibit exceptional resistance to common disinfectants, yet the molecular basis of surfactant-mediated viral inactivation remains poorly understood. Sodium dodecyl sulfate (SDS) demonstrates broad-spectrum virucidal activity, but how it disrupts robust protein capsids and how environmental pH modulates this process is unclear. Here, we report a multiscale simulation framework combining coarse-grained and atomic-resolution icosahedral "scaffold" models to elucidate SDS-driven disruption of the MS2 bacteriophage capsid. Dynamic light scattering and transmission electron microscopy confirm that SDS inactivates MS2 in a strongly pH-dependent manner, triggering capsid disassembly at acidic pH while leaving particles largely intact at neutral pH. Simulations reveal that, under acidic conditions, protonation of acidic residues weakens the capsid's electrostatic network, enabling SDS micelles to preferentially target icosahedral pores and inter-dimer clefts. These findings provide a molecular framework for SDS virucidal action and offer a foundation for designing next-generation antiviral surfactants with improved efficacy and biocompatibility.

Immunoglobulin G (IgG) monoclonal antibodies dominate current cancer immunotherapy but face challenges including resistance development, limited tumor penetration, and suboptimal avidity. In contrast, the pentameric or hexameric architecture of immunoglobulin M (IgM) offers up to twelve antigen-binding sites and potent complement activation, positioning IgM as a promising next-generation therapeutic scaffold. Here, we present integrative structural modeling and multiscale molecular dynamics simulations of IgM versions of Cetuximab and Matuzumab targeting the epidermal growth factor receptor (EGFR), a clinically validated oncogenic driver. Our analyses reveal that IgM antibodies maintain a rigid, glycan-stabilized Fc core while their Fab domains exhibit high mobility, enabling multivalent EGFR binding. Compared with IgG, IgM antibodies demonstrated enhanced binding avidity, prolonged receptor engagement, and slower dissociation kinetics. These properties suggest superior therapeutic durability and potential to overcome current limitations of IgG-based therapies. By providing mechanistic insight into how IgM isotypes can improve therapeutic engagement with tumor-associated antigens, our study supports the development of IgM antibodies as a new class of cancer immunotherapies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis is shaped not only by viral entry mechanisms but also by interactions with host and microbial factors. The viral spike (S) protein can bind Gram-negative bacterial lipopolysaccharide (LPS), a driver of hyperinflammation in severe COVID-19. How viral evolution over the years alters this interaction remains unclear. Here, we investigated LPS binding across major SARS-CoV-2 variants that emerged over the course of the pandemic (2019-2023) from the ancestral Wuhan-Hu-1 strain to Omicron subvariants BA.1, XBB.1.5, and BA.2.86. Structural mapping revealed multiple mutations near a cryptic lipid-binding pocket in the receptor binding domain (RBD). Using extensive atomic-resolution molecular dynamics (MD) simulations with free energy calculations, validated by biochemical binding assays and fluorescence quenching experiments, we show that these mutations weaken binding to the lipid A component of LPS. However, full-length LPS binds with similar affinity to most variants likely due to increased positive electrostatic potential of the RBD, promoting compensatory interactions with negatively charged LPS inner core sugars. Together, these findings uncover an evolutionary balance that preserves S protein-LPS engagement through distinct molecular mechanisms, suggesting that emerging variants may retain the capacity to potentiate hyperinflammation during infection.

Viral replication necessitates intricate nucleic acid rearrangements, including annealing and strand displacement to achieve the viral RNA functional structure. Often a single RNA chaperone performs these seemingly incompatible functions. This raises the question of what structural and dynamic features of such chaperones govern distinct RNA rearrangements. While cationic intrinsically disordered regions promote annealing by playing a charge-screening role, how the same chaperone mediates strand displacement remains elusive. Here, we investigate the annealing and strand displacement of the 5' upstream AUG region (5UAR) as chaperoned by the Dengue virus strain 2 capsid protein (Denv2C) as a model RNA chaperone. Through single molecule analysis and molecular simulations, we demonstrate that Denv2C regulates nucleic acid melting, folding, annealing, and strand displacement via flexibility in its ordered region. A mutation that renders the Denv2C ordered region rigid, converts Denv2C into a mere annealer. Our findings underscore the role of Denv2C's disordered region as a "macromolecular counterion" during RNA annealing, while a flexible ordered region is crucial for effective strand displacement.

Sepsis remains a major clinical challenge due to the limited efficacy of existing therapies in controlling excessive inflammation. The engineered stapled peptide sHVF18, derived from an evolutionarily conserved thrombin innate fold, binds both lipopolysaccharide (LPS) and the LPS-binding groove of CD14, enabling dual targeting of bacterial components and host immune signaling. To define structural prerequisites for this dual action, we combined evolutionary analysis, in silico modeling, and experimental methods. Substituting the N-terminal histidine with lysine (K) or arginine (R) improved solubility, reduced aggregation, and enhanced interactions with LPS. However, unexpectedly, K substitutions impaired CD14 binding, whereas R variants retained weaker affinity, possibly through cation-π interactions. The essential role of the evolutionarily conserved N-terminal histidine for CD14 interactions and therapeutic efficacy was demonstrated using LPS-induced shock and polymicrobial sepsis models. While the K variant exhibited superior efficacy in LPS-induced shock, its disrupted CD14 interactions rendered it ineffective in polymicrobial sepsis. In contrast, sHVF18, by engaging both LPS and CD14, effectively reduced inflammation and improved survival in polymicrobial sepsis. These findings highlight that targeting of both LPS and CD14 is essential for therapeutic efficacy, underscoring multivalency as a key principle for future sHVF18-based sepsis therapeutics.

The MS2 bacteriophage capsid serves as a model system for studying viral structure and function. Mature MS2 virus consists of 178 capsid proteins and a single maturation protein (MP), which is essential for host receptor binding and infection initiation. Despite its critical role, the dynamic behavior of the capsid with the MP remains poorly understood. To address this, we conducted 0.5 µs all-atom molecular dynamics (MD) simulations of the MS2 capsid with and without the MP, revealing key insights into its structural dynamics. Our simulations showed that MP exhibits high flexibility, particularly in the “tip” and “side-loop” regions, which undergo significant motions that likely enhance its ability to engage with the F-pilus receptor. Detailed analysis of MP conformational states revealed that loop rearrangements around H357 enable transient switching between “semi-closed” and “open” conformations, suggesting a conformational selection mechanism for pilus binding. Additionally, ion interaction analyses revealed distinct sodium and chloride binding patterns, where sodium ions were mostly found at the outer capsid shell, while chloride ions interacted with the basic residues on the RNA-facing side. We also found that the presence of the MP enhances salt-bridge interactions, contributing to increased capsid stability, yet it does not significantly alter the pore sizes of pentameric and hexameric units. Together, these findings provide new insights into the functional role of the MP, highlighting its contribution to capsid stability and host receptor engagement. This study offers a foundation for understanding capsid dynamics relevant to viral infectivity and may guide future rational strategies aimed at disrupting host-virus interactions.

Virus-like particles (VLPs) are promising vaccine candidates due to their noninfectious and highly immunogenic nature. Enveloped VLPs are unstable and heterogeneous in size because they lack a viral genomic core, yet they can induce a robust immune response. This motivates the characterization of their biophysical and structural properties to enable rational design of stable, highly immunogenic particles. We employed an integrative approach combining multiscale modeling, structural analyses, and in vitro experiments to gain molecular insights into the factors governing VLP stability, homogeneity, secretion, and antibody binding. We focused on dengue virus VLPs, which elicit neutralizing antibodies similar to infectious virions. Systematic introduction of mutations facing lipid tails in the stem helix of the chimeric E protein, guided by molecular simulations, allowed modulation of secretion efficiency and immunogenicity. Overall, this work highlights the role of envelope protein-lipid interactions in maintaining VLP stability and yield, guiding engineering of improved VLP-based vaccines.

Apolipoprotein E (APOE) is distributed across various human tissues and plays a crucial role in lipid metabolism. Recent investigations have uncovered an additional facet of APOE's functionality, revealing its role in host defense against bacterial infections. To assess the antibacterial attributes of APOE3 and APOE4, we conducted antibacterial assays using Pseudomonas aeruginosa and Escherichia coli. Exploring the interaction between APOE isoforms and lipopolysaccharides (LPSs) from E. coli, we conducted several experiments, including gel shift assays, CD, and fluorescence spectroscopy. Furthermore, the interaction between APOE isoforms and LPS was further substantiated through atomic resolution molecular dynamics simulations. The presence of LPS induced the aggregation of APOE isoforms, a phenomenon confirmed through specific amyloid staining, as well as fluorescence and electron microscopy. The scavenging effects of APOE3/4 isoforms were studied through both in vitro and in vivo experiments. In summary, our study established that APOE isoforms exhibit binding to LPS, with a more pronounced affinity and complex formation observed for APOE4 compared with APOE3. Furthermore, our data suggest that APOE isoforms neutralize LPS through aggregation, leading to a reduction of local inflammation in experimental animal models. In addition, both isoforms demonstrated inhibitory effects on the growth of P. aeruginosa and E. coli. These findings provide new insights into the multifunctionality of APOE in the human body, particularly its role in innate immunity during bacterial infections.

The lack of efficacious vaccines against dengue (DENV) infections imposes an enormous burden on global health and the economy. Virus-like particles (VLPs), such as mature DENV VLPs (mDVLPs), have been shown to induce broadly neutralizing antibodies, making them promising next-generation vaccine candidates. However, the limited structural details have restricted efforts to engineer VLPs to attain the desired biophysical and immunological properties. In the current work, we present the cryo-electron microscopy (cryo-EM) structure of immature dengue serotype 2 VLP (imD2VLPs), revealing an architecture composed of a glycoprotein layer with prominent spikes in a T=1 arrangement. These spikes, composed of envelope (E) and precursor membrane (prM) protein heterodimers capped by pr domains, closely resemble immature flavivirus particles. Complementing the static structural details, we performed multiscale molecular dynamics (MD) simulations to elucidate the functional dynamics of imD2VLPs in the context of different lipid envelope compositions. Additionally, MD simulations uncovered the transition pathway between our previously solved mature VLP structure and immature VLP from this work. Here we show that VLP maturation involves a simple sliding-rotating motion without any clashes between E proteins. Our results also indicated that lipid composition plays a critical role in VLP stability, with phospholipid-dominant environments providing greater stability than diacylglycerol-rich vesicles. In addition, we demonstrated enhanced production efficiency of VLPs by generating a stable mammalian cell line using CHO-K1 cells. These findings enabled us not only to predict and manipulate the immunogenic properties of dengue VLPs but also underscored the potential of VLPs as a simplified and manageable model for investigating the structural basis of the dengue virus more effectively. ### Competing Interest Statement The authors have declared no competing interest.

The envelope (E) protein of dengue virus (DENV) is glycosylated at two highly conserved asparagine (N) sites (N67 and N153). The role and importance of these N-linked glycans in DENV pathogenesis has been elusive. Here, we report the critical role of N153-linked glycans on E protein in preventing antibody-mediated viral clearance. A DENV2 mutant lacking N153-linked glycans (N153Q mutant) was engineered and found to be mildly impaired in vitro but drastically attenuated in a symptomatic mouse model of severe dengue, as evidenced by accelerated viral clearance. In B cell-deficient mouse models, N153Q mutant displayed parental virulence and viremia profile. Homologous and heterologous passive transfers of purified IgM from infected B cell-proficient mice into B cell-deficient mice demonstrated the role of N153Q-specific IgM in N153Q attenuation and accelerated clearance, while WT DENV was unaffected by IgM from both WT- and N153Q-infected mice. Furthermore, in vitro neutralization assay supported that the accelerated clearance of N153Q mutant in mice was mediated by non-neutralizing IgM. Furthermore, using plasma samples from convalescent dengue patients and monoclonal antibodies, in vitro neutralization assays showed that N153Q virus was more susceptible than WT to IgG-mediated neutralization. Glycoproteomics combined with molecular dynamics (MD) simulations revealed that glycan composition on E protein influenced IgG binding. Our findings were extended to all DENV serotypes and ZIKV, hence supporting that the N153 glycans-mediated immune evasion strategy is conserved across orthoflaviviruses. ### Competing Interest Statement The authors have declared no competing interest.

Extrinsic apoptosis is initiated by signaling from death receptors, leading to the assembly of RIPK1, FADD, and caspase-8 complex. Subsequently, caspase-8 forms a filamentous structure through the oligomerization of its tandem death effector domain (tDED), resulting in caspase activation and cell death. Although the DED of FADD (FADDDED) is homologous to the tDEDs of caspase-8 (casp8tDED) and both oligomerize to function, the functional form of FADDDED oligomer in extrinsic apoptosis remains unclear. Here, using cryogenic-electron microscopy, we elucidate the structure of FADDDED filaments comprising three helical chains assembled through three types of iterative interactions. Mutations disrupting FADDDED filament formation impair the recruitment of RIPK1 and caspase-8, and abrogate the cell death response, suggesting that FADDDED filamentation represents an important mechanistic step in the initiation of TNF-induced extrinsic apoptosis. Contrary to the belief that the homotypic death domains of RIPK1 and FADD are solely responsible for their interaction, we here show this interaction requires FADDDED filamentation. Furthermore, cFLIP can disrupt FADDDED filaments, uncovering an additional antiapoptotic mechanism of cFLIP beyond its disruption of caspase-8 filament. Molecular dynamics simulations reveal that FADDDED filament thermodynamically favors casp8tDED monomer over FADDDED monomer, thus explaining the hierarchy and stoichiometry of FADD/caspase-8 complex assembly. These findings highlight the hitherto unappreciated roles of FADDDED filament formation in extrinsic apoptosis.

Dengue virus infection remains a public health threat. Dengue NS2B-NS3 proteins are prime antiviral drug targets, highly dynamic, and adopt different structural conformations. We combine cross-linking mass spectrometry (XL-MS), molecular dynamics (MD) simulations, and biochemical assays to identify NS2B-NS3 full length interactions. Using cross-linkers of different lengths as molecular rulers, we identified NS2B S48 as a key interacting residue with NS3 by XL-MS. Structural modeling with MD simulations revealed a novel compact conformation of the NS2B-NS3 complex. Mutation of NS2B S48 to alanine or lysine greatly reduced protease activity and disrupted the binding pocket in MD simulations with a loss of NS2B-NS3 interactions. Additionally, NS2B-NS3 cross-links were found to be conserved across all four dengue serotypes. Our interdisciplinary approach reveals a new key interacting residue and a compact conformation that are structurally and functionally important for the dynamic NS2B-NS3 complex. These results can help guide drug development against dengue.

Non-structural protein 1 (NS1) of dengue virus (DENV) harbours two conserved N-glycosylation sites at positions 130 and 207, whose biological roles have remained elusive. Using a clinically relevant mouse model of severe dengue, we showed that DENV that lacked N207 glycans on NS1 was significantly attenuated, and this phenotype was dominant over wild-type virulent DENV. Mice infected with this mutant exhibited accelerated viral clearance, milder lymphopenia and more functional DENV-specific CD8+ T cells. Bulk and single-cell RNA sequencing, cytokine measurements and immune-phenotyping revealed blunted innate inflammatory responses early post-infection, which correlated with reduced PD-L1 expression on innate immune cells and reduced PD-1+ T-cells in mice infected with de-glycosylated DENV. PD-1 blockade demonstrated the involvement of premature T-cell apoptosis through the PD-L1/PD-1 axis in DENV pathogenesis. Collectively, our findings support that N207-de-glycosylated NS1 inhibits early inflammatory responses, which restricts PD-L1 upregulation on innate immune cells, which in turn limits PD-L1/PD-1 mediated T-cell apoptosis. Our study uncovers a novel immune evasion strategy and identifies PD-L1/PD-1 as a novel mechanism of dengue immunopathogenesis.

Pathogen recognition by the immune system relies on germline-encoded pathogen recognition receptors which identify conserved pathogen-associated molecular patterns (PAMPs) such as the lipid A section of the lipopolysaccharide (LPS). The assumption that pathogens and mammalian-associated bacteria remodel their lipid A PAMP because of host-microbe co-evolution is a long held-belief of microbial pathogenesis. We set out to test this fundamental principle by interrogating a Gram-negative genus presenting evidence of evolutionary events linked to the acquisition of essential virulence traits, resulting in pathogenic and non-pathogenic species. The genus Yersinia fulfil these requirements; the acquisition of the pYV virulence plasmid is one of the evolutionary events associated with virulence. At 37C, only pathogenic Yersinia switch to deacylated lipid A, a modification that diminishes TLR4/MD-2 recognition and reduces inflammation. An engineered chimeric pathogenic Yersinia strain expressing the non-pathogenic lipid A profile efficiently engages with TLR4, demonstrating it is sufficient to switch the acylation pattern to modify the recognition by TLR4 and subsequent activation of inflammation. The lipid As of pathogenic and non-pathogenic species are modified with aminoarabinose and palmitate; therefore, only the reduced acylation of the lipid A PAMP is a trait associated with virulence. The decorations of lipid A do not alter TLR4 engagement but confer resistance to antimicrobial peptides. The chimeric pathogenic Yersinia strain expressing the non-pathogenic lipid A profile allows to ascertain whether the switch in the lipid A PAMP affects virulence. This strain showed enhanced motility due to an upregulation of the flhDC master regulator, and impaired cellular invasion through downregulation of rovA, a key invasion regulator. The expression and function of pYV-encoded virulence factors Yops and YadA were not affected. Nonetheless, the chimeric strain was attenuated in vivo, demonstrating that virulence factors cannot overcome a switch in the lipid A PAMP associated with pathogenicity. ### Competing Interest Statement J.A.B. declares concultancy fees from VaxDyn adn GSK.

The Acinetobacter baumannii F1FO-ATP synthase is essential for the opportunistic human pathogen. Its membrane-embedded FO domain consists of the c-ring and subunit a. The c-ring translocates protons via a conserved carboxylate across the membrane via two half-channels in subunit a, and its revolution enables the F1 domain to carry out ATP formation. Here, we used molecular dynamics simulations, free energy calculations, and in vivo mutational experiments to assess the likely existence of water molecules in the binding site of the A. baumannii c-ring. We first predicted its binding site structure in the ion-locked conformation and extrapolated the presence of two water molecules in the ion-binding site. Based on our predictions, amino acid point mutations confirmed the critical role of key residues involved in the water-binding site upon ATP synthesis ability and cell growth. We discuss the implications of our findings in the context of rational drug design to target the A. baumannii FO domain.

The F1FO-ATP synthase engine is essential for viability and growth of non-tuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the FO-domain of the engine and preventing rotation and proton-translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307-tebipenem-avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.

The non-structural protein 1 (NS1) of dengue virus (DENV) contains two highly conserved N-glycosylation sites at positions 130 and 207 (N130 and N207). Intracellular NS1 monomers and homo-dimers participate in viral RNA replication within membrane-bound replication complexes. Soluble multimeric NS1 (sNS1) is secreted into the extracellular milieu and represents an important virulence factor for DENV through its ability to interfere with the host complement activation cascade and to induce vascular leakage. The role of the two N-glycans in NS1 biological activities, however, has not been carefully examined. Here, stable DENV2 mutants that lack glycan at either N sites of NS1 were engineered. We showed that the lack of glycans at either N site of NS1 did not impair viral replication nor viral output in both mosquito and mammalian cell lines. In contrast, while N130 de-glycosylated DENV displayed parental in vivo fitness in IFNAR-/- mice, the N207 de-glycosylated mutant was significantly attenuated as evidenced by 100% survival rate, which correlated with accelerated viral clearance in circulation. sNS1-depletion, sNS1 exogenous administration and co-infection experiments supported that N207 de-glycosylated NS1 exerted a dominant attenuating effect during in vivo infection. Bulk RNAseq, inflammatory cytokine profile, immune phenotyping of neutrophils and T cells, immune cell depletion and immune checkpoint blockade approaches led us to propose that N207 de-glycosylated NS1 limited CD8+ T cell apoptosis mediated by the PD-L1/PD-1 axis, thereby improving viral clearance efficacy. This work uncovers a novel immune evasion strategy where N207 glycans on NS1 prevent the protein from exerting immune modulation activity that would be detrimental to DENV.### Competing Interest StatementThe authors have declared no competing interest.

Proteoglycans contain glycosaminoglycans (GAGs) which are negatively charged linear polymers made of repeating disaccharide units of uronic acid and hexosamine units. They play vital roles in numerous physiological and pathological processes, particularly in governing cellular communication and attachment. Depending on their sulfonation state, acetylation, and glycosidic linkages, GAGs belong to different families. The high molecular weight, heterogeneity, and flexibility of GAGs hamper their characterization at atomic resolution, but this may be circumvented via coarse-grained (CG) approaches. In this work, we report a CG model for a library of common GAG types in their isolated or proteoglycan-linked states compatible with version 2.2 (v2.2) of the widely popular CG Martini force field. The model reproduces conformational and thermodynamic properties for a wide variety of GAGs, as well as matching structural and binding data for selected proteoglycan test systems. The parameters developed here may thus be employed to study a range of GAG-containing biomolecular systems, thereby benefiting from the efficiency and broad applicability of the Martini framework.