The clinical efficacy of systemic oncolytic virotherapy (OV) is constrained by the rapid development of neutralizing antibodies (nAbs), which prevent repeat systemic administration, a critical barrier to sustained anti-tumor immunity. Vesiculoviruses offer potent oncolytic and immunogenic potential. However, leveraging their serological diversity for repeat dosing remains unexplored. We generated a library of chimeric vesiculovirus vectors incorporating glycoproteins from less well characterized vesiculovirus species. We evaluated vector replication, infectivity, interferon (IFN) responses, and oncolysis in vitro, alongside assessments of neutralization resistance using patient sera, monoclonal antibodies, and in silico structural modeling. In vivo studies assessed tumor delivery, immune activation, and therapeutic efficacy following intravenous administration. The vesiculovirus library exhibited broad tumor infectivity, distinct IFN-stimulatory profiles, and variable oncolytic activity. Neutralization assays and computational modeling identified serological distinctness across vectors, driven by hypervariable glycoprotein epitopes, enabling evasion of cross-neutralizing antibodies. Tumor delivery and anti-tumor immunity were preserved despite humoral barriers. Incorporating tumor-associated antigens (TAAs) further amplified anti-tumor responses, even in the context of anti-viral memory. Sequential administration of distinct vesiculovirus vectors induced robust immune activation and improved survival in a B16-OVA-IFNAR-/- model. Our findings establish a glycoprotein-diverse vesiculovirus platform capable of overcoming humoral immunity, enabling repeat intravenous dosing and sustained engagement of the tumor microenvironment. This strategy advances the field of oncolytic virotherapy by addressing a major translational barrier and lays the groundwork for future clinical studies integrating multi-vector, multi-dose immunovirotherapy with immune checkpoint blockade.

Wood that is resistant to fungal degradation is highly desirable, and chemical acetylation is one way to inhibit wood decay. The open question is whether acetylation inhibits decay strictly by reducing moisture content within the cell wall or if specific interactions with the acetyl group hinder motion within the cell wall and further inhibit decay. We investigate these hypotheses through molecular simulation, acetylating exposed hemicellulose and lignin hydroxyl groups in a secondary plant cell wall model. By comparing diffusion within the model, we find that acetyl group interactions alone do not account for reduced transport, with only modest changes in diffusion when water saturated acetylated cell walls expand at a constant water content. The most substantial changes in diffusion occur where the additional acetylation displaces water, decreasing the moisture content for the cell wall. These findings elucidate the molecular mechanism through which acetylation affects secondary plant cell walls at atomic resolution.

Bacterial microcompartments (BMCs) are protein shells encapsulating multiple enzymes of a metabolic pathway. Interpretations of early experiments on carboxysomes led to the narrative that transport of small gases (CO2, O2) across the shell membrane is restricted. Since then, this notion has been largely contradicted by studies of engineered shells, although these shell constructs lack important proteins present in the native BMCs, altering the synthetic shells' topology, surface and mechanical properties. We discuss here an updated model of gas permeability that informs the design of engineered shells for catalysis on gas substrates and outline how nonshell suprastructures of BMC shell proteins could be used in formulating sustainable biomaterials for hydrogen generation via methane pyrolysis and for other greenhouse gas mitigations.

Lignin and carbohydrate rich secondary plant cell walls are abundantly available in the biosphere, and is a notable renewable feedstock for biofuels and biomaterials. Particularly for construction applications, wood that is resistant to fungal degradation is highly desirable. Chemical modifications, such as acetylation, have been successfully demonstrated to inhibit wood decay by microorganisms. It is well known that acetylation reduces wood moisture content, which slows down motions within the cell wall when measured by X-ray fluorescence microscopy experiments and molecular simulations. The open question is whether acetylation inhibits decay strictly by reducing moisture content, or if specific interactions with the acetyl group hinder motion within the cell wall and further inhibit decay. We investigate these hypotheses directly through molecular simulation, acetylating exposed hemicellulose and lignin hydroxyl groups in existing models for secondary plant cell wall structure to 5-18% weight-percent gain. By comparing diffusive behavior for cell wall polymers, water, and select ions (Na+ and Fe3+ ), we can track the dynamics within the cell wall and identify the causal mechanisms for reduced transport and uptake of these metal ions by acetylated cell walls. We find that the change from hydroxyl to acetyl group alone does not account for reduced transport, with only modest changes in diffusion when acetylated cell walls are expanded to provide constant moisture level. The most substantial changes in diffusion occur where the additional acetylation displaces water, reducing the moisture content for the cell wall. Utilizing these simulations, we further analyze the interactions between ions and cell wall polymers and the evolution of dynamic water pockets within the structure. Ions interact more frequently with the acetyl group than the hydroxyl groups they replace, yielding to increased ion interactions on aggregate upon acetylation. Collectively, these findings elucidate the molecular mechanism through which acetylation affects secondary plant cell walls at atomic resolution.

We report the performance of the protein complex prediction approaches of our group and their results in CAPRI Rounds 47-55, excluding the joint CASP Rounds 50 and 54, as well as the special COVID-19 Round 51. Our approaches integrated classical pipelines developed in our group as well as more recently developed deep learning pipelines. In the cases of human group prediction, we surveyed the literature to find information to integrate into the modeling, such as assayed interface residues. In addition to any literature information, generated complex models were selected by a rank aggregation of statistical scoring functions, by generative model confidence, or by expert inspection. In these CAPRI rounds, our human group successfully modeled eight interfaces and achieved the top quality level among the submissions for all of them, including two where no other group did. We note that components of our modeling pipelines have become increasingly unified within deep learning approaches. Finally, we discuss several case studies that illustrate successful and unsuccessful modeling using our approaches.

Bacterial microcompartments are made from proteins that form shells to encapsulate specific enzymatic pathways. By spatially sequestering sequential enzymes within a semi-permeable shell proteins, bacterial microcompartments create an environment to efficiently catalyze complex reactions. The shell proteins have hexagonal symmetry, and so the shell proteins can form isocsahedral shells as well as cylinder or sheets. Our primary interest is to determine how permeability or pore shape changes as the protein shell adopts these different overall morphologies. Our initial focus is describing the difference between a curved/capsular shell protein assembly and a shell protein plane. We use molecular modeling tools to create atomically detailed models for both protein sheets and curved structures, and then animated these structures using classical molecular dynamics simulations. From the resulting trajectories, we analyzed overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric proteins from Haliangium ochraceum that form the microcompartment. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. With little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability. The sheet-like planar morphology enables smaller simulations by creating a planar grid to create a membrane-like environment for further study, including explicit permeation calculations.

A comprehensive understanding of lipid structure and dynamics is essential to understanding biomembrane functions. Based on the flexible surface model (FSM), we highlight the significance of proteolipid interactions and their out-of-plane couplings, which influence membrane functionality through curvature elastic forces. These forces, underscored by spontaneous curvature and bending rigidity, are paramount to distinguishing the FSM from the earlier fluid-mosaic model. Investigating membrane mimics consisting of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) may contribute to addressing this question [1]. Here we used solid-state deuterium NMR spectroscopy to investigate the structural dynamics of unsaturated phospholipid mixtures with varying PC to PE headgroups [2]. Using POPC-d31 as a probe lipid, we analyzed the structural and mechanical properties of 1,2-dioleoy-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and their binary mixtures. Measurements were performed on liquid-crystalline state multilamellar dispersions as a function of temperature. The order parameter (SCD) profiles indicated that the quadrupolar splitting remains constant across a probe lipid concentration of 1–15 mol% for both PE and PC lipids, while the mol% of POPC-d31 influences the transition from lamellar to hexagonal phase in DOPE lipids. In the liquid-crystalline phase, DOPE lipids exhibited a constrained acyl chain mobility, leading to a more pronounced molecular order than DOPC. Increasing DOPE concentration reduced the area per molecule at the lipid/water interface, affecting acyl chain configurational freedom and increasing order. Additionally, DOPE membranes showed higher bending rigidity than DOPC, highlighting it is driven primarily by increased lipid packing, correlating with the area compressibility modulus. The use of a probe lipid elucidates the intricate properties of complex lipid systems, facilitating a deeper understanding of how membrane mechanics are intertwined with critical biological functions.

As global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand enzyme features underlying the thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold-predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation-guided structure-based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates.

Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria which encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semi-permeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles; hexamers, trimers, and pentamers, and these tiles self-assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo, the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We use molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo. Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceum model BMC shell. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.

AbstractAntifungal echinocandins inhibit the biosynthesis of β−1,3-glucan, a major and essential polysaccharide component of the fungal cell wall. However, the efficacy of echinocandins against the pathogen Aspergillus fumigatus is limited. Here, we use solid-state nuclear magnetic resonance (ssNMR) and other techniques to show that echinocandins induce dynamic changes in the assembly of mobile and rigid polymers within the A. fumigatus cell wall. The reduction of β−1,3-glucan induced by echinocandins is accompanied by a concurrent increase in levels of chitin, chitosan, and highly polymorphic α−1,3-glucans, whose physical association with chitin maintains cell wall integrity and modulates water permeability. The rearrangement of the macromolecular network is dynamic and controls the permeability and circulation of the drug throughout the cell wall. Thus, our results indicate that echinocandin treatment triggers compensatory rearrangements in the cell wall that may help A. fumigatus to tolerate the drugs’ antifungal effects.

Bacterial microcompartments (BMC), such as carboxysomes, are organelles found in cyanobacteria that locally concentrate carbon dioxide (CO2) to improve the efficiency of the enzyme RuBisCO, a key step of photosynthetic carbon fixation in the Calvin-Benson-Bassham cycle. The carboxysome shell is comprised of shell proteins and encapsulates RuBisCO and carbonic anhydrase. Carbonic anhydrase converts soluble bicarbonate to CO2, increasing the local concentration of CO2 for RuBisCO. In addition to CO2, another metabolite ribulose-1,5-bisphosphate (RuBP) also need to permeate through the carboxysome shell to efficiently perform the CO2 fixation step.

The modeling of diffusion processes, particularly in confined or crowded environments, is considered one of the perennial challenges of classical molecular dynamics or MD simulations. By making assumptions of a friction-dominated regime, Brownian dynamics or BD methods offer an elegant computational solution for studying biomolecular diffusion at the milliseconds timescale, still maintaining the pairwise interactions of MD—a tool dubbed atom resolved BD or ARBD. It is nontrivial to conceive such simulations, given requirements to (1) periodically parametrize BD when MD visits novel conformations, (2) reset pH and charge environment, and (3) update visualization and analysis scripts for the redefined Brownian particles.

Carboxysomes are protein microcompartments found in cyanobac- teria, whose shell encapsulates rubisco at the heart of carbon fixa- tion in the Calvin-Benson-Bassham (CBB) cycle. Carboxysomes are thought to locally concentrate CO2 and exclude O2 from the shell interior to improve rubisco efficiency through selective metabolite permeability, creating a concentrated catalytic center. However, per- meability coefficients have not previously been determined for these gases, or for CBB cycle intermediates such as bicarbonate (HCO3 – ), 3-phosphoglyceric acid (3-PGA), or ribulose-1,5-bisphosphate (RuBP). Starting from a high resolution cryo-EM structure of a synthetic β-carboxysome shell, we perform unbiased all-atom molecular dynamics (MD) to track metabolite permeability across the shell. The synthetic carboxysome shell is found to have similar permeability coefficients for multiple metabolites, and is not selectively permeable to HCO3– relative to CO2. To resolve how these comparable permeabilities can be reconciled with the clear role of the carboxysome in the carbon-concentrating mechanism in cyanobacteria, complementary atomic-resolution Brownian Dynamics (ARBD) simulations estimate the mean first passage time for CO2 assimilation in a crowded model carboxysome. Despite a relatively high CO2 permeability of 10−2 cm/s across the carboxysome shell, the shell proteins reflect enough CO2 back towards rubisco that over 2600 CO2 molecules can be fixed by rubisco for every 1 CO2 molecule that escapes under typical conditions. The permeabilities determined from all-atom molecular simulation are key inputs into flux modeling, and the insight gained into carbon fixation can facilitate the engineering of carboxysomes and other bacterial microcompartments for multiple applications.

Ferredoxins (Fds) are iron-sulfur cluster proteins found in all organisms from bacteria, archaea to higher eukaryotes and function as electron carriers between donor and acceptor pairs. In Chlamydomonas reinhardtii, nine distinct Fd isoforms are localized within the chloroplast which are implicated in photosynthetic electron transport, potentially exhibiting specificity for particular donor-acceptor pairs. Employing all-atom molecular simulations, we ranked these Fd isoforms based on their binding affinities and stability to Photosystem I (PSI), the electron donor in photosynthesis.