Biology subjects

Marzinek, J. K.

Publications and source records attributed to Marzinek, J. K..

15 recordsLinked to original sources

bioRxiv2025

A Multiscale Framework for Uncovering Surfactant Mediated Viral Capsid Disruption

Disinfection remains a critical strategy for controlling the transmission of infectious diseases. However, small non-enveloped viruses exhibit exceptional resistance to many disinfectants, often requiring harsh protein-disrupting chemicals for effective inactivation, thereby limiting their applicability in personal care products due to associated side effects. Sodium dodecyl sulphate (SDS) is a widely used anionic surfactant known for its virucidal efficacy; however, the molecular details of its action against robust non-enveloped viruses remain poorly understood, limiting efforts to design safer and more targeted antiviral formulations. In this study, a multiscale simulation approach combining a novel atomic-resolution icosahedral "scaffold framework" and coarse-grained modelling was developed to elucidate the mechanism of SDS-driven disruption of MS2 bacteriophage capsid, a surrogate for non-enveloped viruses. Experimental analyses including dynamic light scattering and transmission electron microscopy revealed that SDS inactivates MS2 in a strongly pH-dependent manner, triggering capsid disassembly at acidic pH while leaving particles largely intact at neutral pH. Molecular dynamics simulations demonstrated that SDS micelles preferentially associate with hexameric pores and inter-dimer clefts under acidic conditions, where protonation of acidic residues weakens the electrostatic network of the capsid surface. Together, these findings provide a detailed molecular framework for SDS virucidal action and highlight the importance of environmental pH in modulating surfactant-virus interactions. These insights offer a foundation for designing next-generation antiviral surfactants with improved efficacy and biocompatibility.

biophysics↗

bioRxiv2025

An Integrative Approach to Rational Engineering of Dengue Virus-Like Particles

Virus-like particles (VLPs) are promising vaccine candidates due to their noninfectious and highly immunogenic nature. These particles lack a viral genomic core and display a robust host immune response. VLPs are typically highly unstable and heterogeneous in size. This motivates the characterization of the biophysical and structural properties of VLPs to facilitate the rational design of stable and highly immunogenic particles. We employed an integrative approach combining multiscale modeling, lipidomics, and in vitro experiments to gain molecular insights into the factors governing VLP stability and homogeneity. We focused on dengue virus VLPs, which are known to elicit neutralizing antibodies similar to infectious virions. Systematic introduction of mutations in the structurally crucial stem helix region of the chimeric E protein guided by molecular simulations allowed us to modulate the secretion efficiency of VLPs in vitro. Overall, this work highlights the role of protein{square}lipid envelope interactions in maintaining VLP stability with better yield, providing a framework for the future development of stable and immunogenic next-generation VLP-based vaccines.

biophysics↗

bioRxiv2025

Interrogating the genus Yersinia to define the rules of lipopolysaccharide lipid A structure associated with pathogenicity.

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 37{degrees}C, 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.

microbiology↗

bioRxiv2025

N153-linked glycans on envelope protein protect orthoflaviviruses from antibody-mediated clearance

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.

microbiology↗

bioRxiv2025

Structural Insights into Immature Dengue Virus-Like Particles Revealed by Cryo-EM and Molecular Dynamics Simulations

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.

bioinformatics↗

bioRxiv2024

De-glycosylated non-structural protein 1 enhances dengue virus clearance by limiting PD-L1/PD-1 mediated T cell apoptosis

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.

microbiology↗

bioRxiv2023

A coarse-grained model of glycosaminoglycans for biomolecular simulation

Proteoglycans contain glycosaminoglycans (GAGs), 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 governing cellular communication and attachment. Depending on their sulphonation state, acetylation, and glycosidic linkages, GAGs belong to different families. The high molecular weight, heterogeneity, and flexibility of GAGs hampers 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 the widely popular CG Martini forcefields (versions 2.2 and 3.0). 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, benefitting from the efficiency and broad applicability of the Martini framework.

biophysics↗

bioRxiv2022

Impact on S. aureus and E. coli membranes of treatment with chlorhexidine and alcohol solutions: insights from molecular simulations and nuclear magnetic resonance.

Membranes form the first line of defence of bacteria against potentially harmful molecules in the surrounding environment. Understanding the protective properties of these membranes represents an important step towards development of targeted anti-bacterial agents such as sanitizers. Use of propanol, isopropanol and chlorhexidine can significantly decrease the threat imposed by bacteria in the face of growing anti-bacterial resistance via mechanisms that include membrane disruption. Here we have employed molecular dynamics simulations and nuclear magnetic resonance to explore the impact of chlorhexidine and alcohol on the S. aureus cell membrane, as well as the E. coli inner and outer membranes. We identify how sanitizer components partition into these bacterial membranes, and show that chlorhexidine is instrumental in this process.

biophysics↗

bioRxiv2022

A pH-dependent cluster of charges in a conserved cryptic pocket on flaviviral envelopes

Flaviviruses are enveloped viruses which include numerous human pathogens of escalating global health concern that are predominantly transmitted by mosquitoes and ticks. Some, such as dengue virus, exhibit the phenomenon of antibody-dependent enhancement (ADE) of disease, making traditional vaccine-based routes of fighting viral infections problematic. The pH-dependent conformational change of the envelope (E) protein required for fusion between the viral and endosomal membranes is an attractive point of inhibition by antivirals as it also has the potential to diminish the effects of ADE. Here, we systematically examined six flaviviruses by employing large-scale molecular dynamics (MD) simulations of a [~]400,000 atom raft system that represents a substantial portion of the curved flaviviral envelope. We utilised a benzene-mapping approach over a total of 14 s of sampling time, leading to discovery of shared hotspots and elucidation of the dynamic behaviour of conserved cryptic sites. A cryptic pocket previously shown to bind a detergent molecule exhibited significant strain-specific characteristics. An alternative conserved cryptic site located at the E protein domain interfaces showed a more consistent dynamic behaviour across flaviviruses and contains a buried, conserved cluster of ionisable residues that includes His144, previously implicated in the pH-dependent conformational switch required for fusion. The dynamics of the cluster were further explored in extensive constant-pH simulations and revealed cluster and domain-interface disruption under low pH conditions. Based on this, we propose a cluster-dependent mechanism that addresses inconsistencies in the histidine-switch hypothesis and highlights the role of cluster protonation in orchestrating the domain dissociation pivotal for the formation of the fusogenic trimer. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/499710v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@2a90feorg.highwire.dtl.DTLVardef@9535a1org.highwire.dtl.DTLVardef@1f41ae0org.highwire.dtl.DTLVardef@1106e41_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Viral genome-capsid core senses host environments to prime RNA release

Viruses are metastable macromolecular assemblies containing a nucleic acid core packaged by capsid proteins that are primed to disassemble in host-specific environments leading to genome release and replication. The mechanism of how viruses sense environmental changes associated with host entry to prime them for disassembly is unknown. We have applied a combination of mass spectrometry, cryo-EM, and simulation-assisted structure refinement to Turnip crinkle virus (TCV), which serves as a model non-enveloped icosahedral virus (Triangulation number = 3, 180 copies/icosahedron). Our results reveal genomic RNA tightly binds a subset of viral coat proteins to form a stable RNA-capsid core which undergoes conformational switching in response to host-specific environmental changes. These changes include: i) Depletion of Ca2+ which triggers viral particle expansion ii) Increase in osmolytes further disrupt interactions of outer coat proteins from the RNA-capsid core to promote complete viral disassembly. A cryo-EM structure of the expanded particle shows that RNA is asymmetrically extruded from a single 5-fold axis during disassembly. The genomic RNA:capsid protein interactions confer metastability to the TCV capsid and drive release of RNA from the disassembling virion within the plant host cell. AUTHOR SUMMARYRNA viruses including coronaviruses, dengue, influenza, and HIV are a significant threat to human health. These viral particles are finely tuned to undergo complex conformational changes that allow for response to varied environments. Turnip crinkle virus (TCV) serves as an excellent model for studying RNA virus dynamics. Since TCV is non-enveloped and has no post-translational modifications, we can specifically investigate the contributions of RNA to viral dynamics. Genomic RNA is not a passive entity but plays a crucial and previously uncharacterized role in viral disassembly. Our results reveal that the genomic RNA-capsid core serves as an environmental sensor and undergoes conformational switching in response to host cell conditions.

plant biology↗

bioRxiv2021

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

The recent global COVID-19 pandemic has prompted a rapid response in terms of vaccine and drug development targeting the viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we modelled a complete membrane-embedded SARS-CoV-2 spike (S) protein, the primary target of vaccine and therapeutics development, based on available structural data and known glycan content. We then used molecular dynamics (MD) simulations to study the system in the presence of benzene probes designed to enhance discovery of cryptic, potentially druggable pockets on the S protein surface. We uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop, which was shown to be involved in the formation of S protein multimers on the viral surface. A marked multi-conformational behaviour of this loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments, supportive of opening and closing dynamics. Interestingly, the pocket is also the site of the D614G mutation, known to be important for SARS-CoV-2 fitness, and within close proximity to mutations in the novel SARS-CoV-2 strains B.1.1.7 and B.1.1.28, both of which are associated with increased transmissibility and severity of infection. The pocket was present in systems emulating both immature and mature glycosylation states, suggesting its druggability may not be dependent upon the stage of virus maturation. Overall, the predominantly hydrophobic nature of the cryptic pocket, its well conserved surface, and proximity to regions of functional relevance in viral assembly and fitness are all promising indicators of its potential for therapeutic targeting. Our method also successfully recapitulated hydrophobic pockets in the receptor binding domain and N-terminal domain associated with detergent or lipid binding in prior cryo-electron microscopy (cryo-EM) studies. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.

biophysics↗

bioRxiv2020

Molecular Dynamics Simulations of Bacterial Outer Membrane Lipid Extraction: Adequate Sampling?

The outer membrane of Gram-negative bacteria is almost exclusively composed of lipopolysaccharide in its outer leaflet, whereas the inner leaflet contains a mixture of phospholipids. Lipopolysaccharide diffuses at least an order of magnitude slower than phospholipids, which can cause issues for molecular dynamics simulations in terms of adequate sampling. Here we test a number of simulation protocols for their ability to achieve convergence with reasonable computational effort using the MARTINI coarse-grained force-field. This is tested in the context both of potential of mean force (PMF) calculations for lipid extraction from membranes, and of lateral mixing within the membrane phase. We find that decoupling the cations that cross-link the lipopolysaccharide headgroups from the extracted lipid during PMF calculations is the best approach to achieve convergence comparable to that for phospholipid extraction. We also show that lateral lipopolysaccharide mixing/sorting is very slow and not readily addressable even with Hamiltonian replica exchange. We discuss why more sorting may be unrealistic for the short (microseconds) timescales we simulate and provide an outlook for future studies of lipopolysaccharide-containing membranes.

biophysics↗

bioRxiv2020

Extending the Martini coarse-grained forcefield to N-glycans

Glycans play a vital role in a large number of cellular processes. Their complex and flexible nature hampers structure-function studies using experimental techniques. Molecular dynamics (MD) simulations can help in understanding dynamic aspects of glycans if the forcefield (FF) parameters used can reproduce key experimentally observed properties. Here, we present optimized coarse-grained (CG) Martini FF parameters for N-glycans, calibrated against experimentally derived binding affinities for lectins. The CG bonded parameters were obtained from atomistic (ATM) simulations for different glycan topologies including high mannose and complex glycans with various branching patterns. In the CG model, additional elastic networks are shown to improve maintenance of the overall conformational distribution. Solvation free energies and octanol-water partition coefficients were also calculated for various n-glycan disaccharide combinations. When using standard Martini non-bonded parameters, we observed that glycans spontaneously aggregated in the solution and required down-scaling of their interactions for reproduction of ATM model radial distribution functions. We also optimised the non-bonded interactions for glycans interacting with seven lectin candidates and show that scaling down the glycan-protein interactions can reproduce free energies obtained from experimental studies. These parameters should be of use in studying the role of glycans in various glycoproteins, carbohydrate binding proteins (CBPs) as well as their complexes, while benefiting from the efficiency of CG sampling.

biophysics↗

bioRxiv2020

A benzene-mapping approach for uncovering cryptic pockets in membrane-bound proteins

Molecular dynamics (MD) simulations in combination with small organic probes present in the solvent have previously been used as a method to reveal cryptic pockets that may not have been identified in experimental structures. We report such a method implemented within the CHARMM forcefield to effectively explore cryptic pockets on the surfaces of membrane-embedded proteins using benzene as a probe molecule. This relies on modified non-bonded parameters in addition to repulsive potentials between membrane lipids and benzene molecules. The method was tested on part of the outer shell of the dengue virus (DENV), for which research into a safe and effective neutralizing antibody or drug molecule is still ongoing. In particular, the envelope (E) protein, associated with the membrane (M) protein, is a lipid membrane-embedded complex which forms a dimer in the mature viral envelope. Solvent mapping was performed for the full, membrane-embedded EM protein complex and compared with similar calculations performed for the isolated, soluble E protein ectodomain dimer in solvent. Ectodomain-only simulations with benzene exhibited unfolding effects not observed in the more physiologically relevant membrane-associated systems. A cryptic pocket which has been experimentally shown to bind n-octyl-{beta}-D-glucoside detergent was consistently revealed in all benzene-containing simulations. The addition of benzene also enhanced the flexibility and hydrophobic exposure of cryptic pockets at a key, functional interface in the E protein, and revealed a novel, potentially druggable pocket that may be targeted to prevent conformational changes associated with viral entry into the cell. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/025163v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@abe537org.highwire.dtl.DTLVardef@155a741org.highwire.dtl.DTLVardef@57bff6org.highwire.dtl.DTLVardef@fbee3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2017

Molecular basis for the maintenance of lipid asymmetry in the outer membrane of Escherichia coli

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides (LPS) and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults. In Escherichia coli, the OmpC-MlaA complex is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How it performs this function is unknown. Here, we define the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. We establish that MlaA sits entirely within the bilayer in complex with OmpC and provides a hydrophilic channel possibly for PL translocation across the OM. Furthermore, we show that flexibility in a hairpin loop adjacent to the channel modulates MlaA activity. Finally, we demonstrate that OmpC plays an active role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antimicrobial drug development.

biochemistry↗