The United Nations have committed to end the epidemics of communicable diseases by 2030 (SDG Target 3.3). In contrast with this ambition, the rise of Multi Drug Resistant (MDR) and Pan Drug Resistant (PDR) bacteria poses a threat of a return to the pre-antibiotic era. It is of high priority to find new therapies that target the ESKAPEE group of pathogens and their drug-resistant strains. Antimicrobial peptides (AMPs) are an emerging class of antibiotics that hold promises of overcoming bacterial resistance by using both novel mechanisms of action as well as targeting already known pathways. The chemical space of AMPs is potentially huge and methodologies allowing the rational exploration of novel structures are highly needed. This review focuses on case studies that give novel insights about the mechanisms of action, resistance and selectivity of some relevant AMPs, exemplifying the importance of microscopic, computational and experimental tools. Particular focus will be devoted to bacterial membranes, and how AMPs can target them while sparing human plasma membranes, in order to become safer drugs. The lessons learned from the literature cases give directions towards the development of AMPs as drug products.

Comment to article published in Proc. Natl. Acad. Sci. U. S. A.: Garaizar, A. et al. 'Toward understanding lipid reorganization in RNA lipid nanoparticles in acidic environments.' Proc. Natl. Acad. Sci. U. S. A. 121, e2404555121 (2024)

Constant pH Molecular Dynamics (CpHMD) simulations represent a cutting-edge computational approach for studying biological systems with remarkable realism. Recent advancements have enhanced the accessibility and efficiency of CpHMD, significantly reducing the performance overhead compared to traditional constant-protonation MD simulations. This chapter guides the reader through the application of CpHMD to investigate the pH-dependent behavior of Cationic Ionizable Lipids (CILs) - a critical component of Lipid Nanoparticles (LNPs), which are among the most promising platforms for drug delivery. LNPs, including those employed in mRNA-based vaccines, played a pivotal role in the global response to the SARS-CoV-2 pandemic, underscoring their potential in modern medicine. The chapter begins with a comprehensive introduction to the fundamental concepts of LNPs and provides a step-by-step protocol for setting up simulations of membranes containing CILs to calculate their apparent pKa. This parameter is crucial for governing the in vivo behavior of LNPs, where precise control is essential to optimize delivery efficiency while minimizing toxicity. By showcasing the ability of CpHMD simulations to unravel the intricate relationship between pH-dependent protonation, membrane structure, and lipid distribution, this chapter highlights their potential to inform the rational design of novel LNP formulations. ### Competing Interest Statement The authors have declared no competing interest.

A widely known property of lipid membranes is their tendency to undergo a separation into disordered (Ld) and ordered (Lo) domains. This impacts the local structure of the membrane relevant for the physical (e.g., enhanced electroporation) and biological (e.g., protein sorting) significance of these regions. The increase in computing power, advancements in simulation software, and more detailed information about the composition of biological membranes shifts the study of these domains into the focus of classical molecular dynamics simulations. In this chapter, we present a versatile yet robust analysis pipeline that can be easily implemented and adapted for a wide range of lipid compositions. It employs Gaussian-based Hidden Markov Models to predict the hidden order states of individual lipids by describing their structure through the area per lipid and the average SCC order parameters per acyl chain. Regions of the membrane with a high correlation between ordered lipids are identified by employing the Getis-Ord local spatial autocorrelation statistic on a Voronoi tessellation of the lipids. As an example, the approach is applied to two distinct systems at a coarse-grained resolution, demonstrating either a strong tendency towards phase separation (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DIPC), cholesterol) or a weak tendency toward phase separation (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PUPC), cholesterol). Explanations of the steps are complemented by coding examples written in Python, providing both a comprehensive understanding and practical guidance for a seamless integration of the workflow into individual projects.

Lipid nanoparticles (LNPs) are essential delivery vehicles in mRNA-based vaccines, with their functionality largely governed by aminolipids. At low pH, protonated aminolipids facilitate efficient mRNA encapsulation, while their deprotonation at physiological pH drives LNP remodeling into a stable spherical structure, potentially aiding mRNA release during endosomal re-protonation and concomitant restructuring at low pH. Our study uncovers the pH-dependent phase transition of the Comirnaty lipid formulation at the molecular scale, highlighting the intricate interplay between lipid reorganization and aminolipid protonation. We observe a substantial shift in the aminolipid pKa, from 7-8 at the LNP surface to below 4 within the hydrophobic LNP core, resulting in an overall apparent pKa of the lipid nanoparticle of 6.3. The pH-dependent protonation significantly affects the aminolipid's localization, with protonated aminolipids enriched in vicinity of the mRNA and at the LNP surface at neutral pH. The lipid monolayer covering LNPs at neutral pH comprises cholesterol (60-65%), helper phospholipids (20-25%), PEGylated lipids (5%), and a mix of protonated and deprotonated aminolipids (5-10%). These findings provide mechanistic insights into aminolipids behavior in LNPs, offering a foundation for optimizing LNP design to enhance stability, tunable pH responses, and therapeutic efficacy. ### Competing Interest Statement The authors have declared no competing interest.

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel β-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell's membrane potential.

Dataset repository - Role of lipid nanodomains for inhibitory FcγRIIb function ============================== The repository contains setup files and parameter files for atomistic molecular dynamics (MD) simulations performed with the GROMACS 2021/2022 series. Modified trajectories for the post-processing analysis are further included. The repositiory corresponds to the following manuscript: Franziska Spiegel, Marius F.W. Trollmann, Sibel Kara, Matthias Pöhnl, Astrid F. Brandner, Falk Nimmerjahn, Anja Lux, Rainer A. Böckmann bioRxiv 2023.05.09.540011; doi: https://doi.org/10.1101/2023.05.09.540011 Corresponding authors: * Rainer A. Böckmann (rainer.boeckmann@fau.de) * Anja Lux (anja.lux@fau.de) ### XTC/TPRs Directories contain: * md.tpr: GROMACS run file for the whole system * md_nonsolvent.tpr: GROMACS run file for the non-solvent selection (Membrane-Protein only) * md_mol_center_dt10ns.xtc: GROMACS trajectories used during post-processing with a slightly larger spacing (10ns instead of 1ns). * topol.top: GROMACS topology file used for simulations * index.ndx: GROMACS index file used for simulations * toppar: Contains CHARMM36 forcefield parameters for lipids, protein and glycans in GROMACS readable format (.itp) * step6.6_equilibration.gro: Input structure of the last equilibration step (see step6.6_equilibration.mdp) that was used as inital structure for the production simulation * last_frame.gro: Output structure of the production simulation The preparation of the raw trajectories for post-processing was done with GROMACS using the commands: >> gmx trjconv -f md.xtc -s md.tpr -pbc mol -center -n md_traj.ndx -dt 1000 -o md_mol_center.xtc >> gmx trjconv -f md_mol_center.xtc -s md_nonsolvent.tpr -o md_mol_center_dt10ns.xtc -dt 10000 The group for centering was the transmembrane domain of the Fcg receptor, as output only the protein and the membrane was chosen. #### Wild type simulations (No Glycans) * wt_replica_0 * wt_replica_1 * wt_replica_2 * wt_replica_3 * wt_replica_4 * wt_replica_5 * wt_replica_6 * wt_replica_7 * wt_replica_8 #### Mutant simulations (No Glycans) * mut_replica_0 * mut_replica_1 * mut_replica_2 #### Wild type simulations (Glycans) * wt_glycan_replica_0 * wt_glycan_replica_1 * wt_glycan_replica_2 * wt_glycan_replica_3 ### Parameters Simulation parameter files (.mdp) are given for each equilibration step and the final production simulation. Initial input files are obtained from the CHARMM-GUI webserver. * simulation_parameters * mut_wt_no_glycans * wt_glycans ### Copyright This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ or send a letter to Creative Commons, PO Box 1866, Mountain View, CA 94042, USA.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

Biological membranes, composed mainly of phospholipids and cholesterol, play a vital role as cellular barriers. They undergo localized reshaping in response to environmental cues and protein interactions, with the energetics of deformations crucial for exerting biological functions. This study investigates the non-universal role of cholesterol on the structure and elasticity of saturated and unsaturated lipid membranes. Our study uncovers a highly cooperative relationship between thermal membrane bending and local cholesterol redistribution, with cholesterol showing a strong preference for the compressed membrane leaflet. Remarkably, in unsaturated membranes, increased cholesterol mobility enhances cooperativity, resulting in membrane softening despite membrane thickening and lipid compression caused by cholesterol. These findings elucidate the intricate interplay between thermodynamic forces and local molecular interactions that govern collective properties of membranes.

The inhibitory Fcγ receptor FcγRIIb is involved in immune regulation and is known to localize to specific regions of the plasma membrane called lipid rafts. Previous studies suggested a link between the altered lateral receptor localization within the plasma membrane and the functional impairment of the FcγRIIb-I232T variant that is associated with systemic lupus erythematosus. Here, we conducted microsecond all-atom molecular dynamics simulations and IgG binding assays to investigate the lipid nano-environment of FcγRIIb monomers and of the FcγRIIb-I232T mutant within a plasma membrane model, the orientation of the FcγRIIb ectodomain, and its accessibility to IgG ligands. In contrast to previously proposed models, our simulations indicated that FcγRIIb does not favor a cholesterol- or a sphingolipid-enriched lipid environment. Interestingly, cholesterol was depleted for all studied FcγRIIb variants within a 2-3 nm environment of the receptor, counteracting the usage of raft terminology for models on receptor functionality. Instead, the receptor interacts with lipids that have poly-unsaturated fatty acyl chains and with (poly-) anionic lipids within the cytosolic membrane leaflet. We also found that FcγRIIb monomers adopt a conformation that is not suitable for binding to its IgG ligand, consistent with a lack of detectable binding of monomeric IgG in experiments on primary immune cells. However, our results propose that multivalent IgG complexes might stabilize FcγRIIb in a binding-competent conformation. We suggest differences in receptor complex formation within the membrane as a plausible cause of the altered membrane localization or clustering and the altered suppressive function of the FcγRIIb-I232T variant.

Simulations of lipid membranes typically make use of periodic boundary conditions to mimic macroscopically sized membranes and allow for comparison to experiments performed e.g. on planar lipid membranes or on unilamellar lipid vesicles. However, the lateral periodicity partly suppresses membrane fluctuations or membrane remodeling, processes that are of particular importance in the study of asymmetric membranes-i.e. membranes with integral or associated proteins and/or asymmetric lipid compositions. Here, we devised a simple albeit powerful lipid bicelle model system that (i) displays similar structural, dynamical, and mechanical properties compared to infinite periodic lipid membrane systems and allows (ii) for the study of asymmetric lipid bilayer systems and (iii) the unperturbed formation of local spontaneous curvature induced by lipids or proteins in molecular dynamics simulations. In addition, the system is characterized by largely unbiased thermal fluctuations as opposed to standard bilayer systems. Application of the bicelle system for an asymmetric lipid composition resembling the plasma membrane reveals that the cholesterol density for a tension-free plasma membrane with a vanishing spontaneous curvature is larger by 28% within the extracellular leaflet compared to the cytosolic leaflet.

Redox signaling and cardiac function are tightly linked. However, it is largely unknown which protein targets are affected by hydrogen peroxide (H 2 O 2 ) in cardiomyocytes that underly impaired inotropic effects during oxidative stress. Here, we combine a chemogenetic mouse model (HyPer-DAO mice) and a redox-proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we demonstrate that increased endogenous production of H 2 O 2 in cardiomyocytes leads to a reversible impairment of cardiac contractility in vivo. Notably, we identify the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch, linking its modification to altered mitochondrial metabolism. Using microsecond molecular dynamics simulations and experiments using cysteine-gene-edited cells reveal that IDH3γ Cys148 and 284 are critically involved in the H 2 O 2 -dependent regulation of IDH3 activity. Our findings provide an unexpected mechanism by which mitochondrial metabolism can be modulated through redox signaling processes.

Crucial for mRNA-based vaccines are the composition, structure, and properties of lipid nanoparticles (LNPs) as their delivery vehicle. Using all-atom molecular dynamics simulations as a computational microscope, we provide an atomistic view of the structure of the Comirnaty vaccine LNP, its molecular organization, physicochemical properties, and insight in its pH-driven phase transition enabling mRNA release at atomistic resolution. At physiological pH, our simulations suggest an oil-like LNP core that is composed of the aminolipid ALC-0315 and cholesterol (ratio 72:28). It is surrounded by a lipid monolayer formed by distearoylphosphatidylcholine, ALC-0315, PEGylated lipids, and cholesterol at a ratio of 22:9:6:63. Protonated aminolipids enveloping mRNA formed inverted micellar structures that provide a shielding and likely protection from environmental factors. In contrast, at low pH, the Comirnaty lipid composition instead spontaneously formed lipid bilayers that display a high degree of elasticity. These pH-dependent lipid phases suggest that a change in pH of the environment upon LNP transfer to the endosome likely acts as trigger for cargo release from the LNP core by turning aminolipids inside out, thereby destabilizing both the LNP shell and the endosomal membrane.

Abstract Redox signaling and cardiac function are tightly linked. Still, it is largely unknown which protein targets are affected by H2O2 in cardiomyocytes that underly the impaired inotropic effects in oxidative stress. Here, we combined a new chemogenetic mouse model (HyPer-DAO mice) and a redox proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we prove that increased endogenous production of H2O2 in cardiomyocytes leads to a reversibly impaired cardiac contractility in vivo. We identified the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch and link its modification to mitochondrial metabolism and glutathione synthesis. Microsecond molecular dynamics simulations combined with experiments using cysteine-gene-edited point mutated cells revealed that IDH3γ Cys148 and 284 are critically involved in H2O2-dependent regulation of IDH3 activity. The oxidative modification of IDH3γ regulates the competition between energy production by TCA cycle flux and cellular anti-oxidant defense by glutathione synthesis.

The (local) curvature of cellular membranes acts as a driving force for the targeting of membrane-associated proteins to specific membrane domains, as well as a sorting mechanism for transmembrane proteins, e.g., by accumulation in regions of matching spontaneous curvature. The latter measure was previously experimentally employed to study the curvature induced by the potassium channel KvAP and by aquaporin AQP0. However, the direction of the reported spontaneous curvature levels as well as the molecular driving forces governing the membrane curvature induced by these integral transmembrane proteins could not be addressed experimentally. Here, using both coarse-grained and atomistic molecular dynamics (MD) simulations, we report induced spontaneous curvature values for the homologous potassium channel Kv 1.2/2.1 Chimera (KvChim) and AQP0 embedded in unrestrained lipid bicelles that are in very good agreement with experiment. Importantly, the direction of curvature could be directly assessed from our simulations: KvChim induces a strong positive membrane curvature (≈0.036 nm-1) whereas AQP0 causes a comparably small negative curvature (≈-0.019 nm-1). Analyses of protein-lipid interactions within the bicelle revealed that the potassium channel shapes the surrounding membrane via structural determinants. Differences in shape of the protein-lipid interface of the voltage-gating domains between the extracellular and cytosolic membrane leaflets induce membrane stress and thereby promote a protein-proximal membrane curvature. In contrast, the water pore AQP0 displayed a high structural stability and an only faint effect on the surrounding membrane environment that is connected to its wedge-like shape.

Dendritic cells (DCs) are professional antigen-presenting cells of the immune system. Upon sensing pathogenic material in their environment, DCs start to mature, which includes cellular processes, such as antigen uptake, processing and presentation, as well as upregulation of costimulatory molecules and cytokine secretion. During maturation, DCs detach from peripheral tissues, migrate to the nearest lymph node, and find their way into the correct position in the net of the lymph node microenvironment to meet and interact with the respective T cells. We hypothesize that the maturation of DCs is well prepared and optimized leading to processes that alter various cellular characteristics from mechanics and metabolism to membrane properties. Here, we investigated the mechanical properties of monocyte-derived dendritic cells (moDCs) using real-time deformability cytometry to measure cytoskeletal changes and found that mature moDCs were stiffer compared to immature moDCs. These cellular changes likely play an important role in the processes of cell migration and T cell activation. As lipids constitute the building blocks of the plasma membrane, which, during maturation, need to adapt to the environment for migration and DC-T cell interaction, we performed an unbiased high-throughput lipidomics screening to identify the lipidome of moDCs. These analyses revealed that the overall lipid composition was significantly changed during moDC maturation, even implying an increase of storage lipids and differences of the relative abundance of membrane lipids upon maturation. Further, metadata analyses demonstrated that lipid changes were associated with the serum low-density lipoprotein (LDL) and cholesterol levels in the blood of the donors. Finally, using lipid packing imaging we found that the membrane of mature moDCs revealed a higher fluidity compared to immature moDCs. This comprehensive and quantitative characterization of maturation associated changes in moDCs sets the stage for improving their use in clinical application.