Piotr Setny

We propose an approach for determining the positioning of membrane-active peptides within a lipid bilayer. It is based on a combination of cryogenic electron microscopy (cryo-EM) with molecular dynamics (MD) simulations. Cryo-EM image intensity profiles across peptide-containing liposome membranes are analyzed by comparing them to synthetic images that are derived from MD trajectories of peptide-membrane systems representing different assumed binding modes. These simulated profiles serve as baseline models, which are then used to classify experimentally obtained images into respective categories. The approach was applied to influenza virus fusion peptides, providing evidence for predominantly transmembrane binding in pure POPC membranes and a transition toward surface-bound configurations upon the addition of cholesterol.

Curvature emerges as a fundamental membrane characteristic crucial for diverse biological processes, including vesicle formation, cell signaling, and membrane trafficking. Increasingly valuable insights into atomistic details governing curvature-dependent membrane properties are provided by computer simulations. Nevertheless, the underlying force field models are conventionally calibrated and tested in relation to experimentally derived parameters of planar bilayers, thereby leaving uncertainties concerning their consistency in reproducing curved lipid systems. In this study we compare the depiction of buckled phosphatidylcholine (POPC) and POPC-cholesterol membranes by four popular force field models. Aside from agreement with respect to general trends in curvature dependence of a number of parameters, we observe a few qualitative differences. Among the most prominent ones is the difference between atomistic and coarse grained force fields in their representation of relative compressibility of the polar headgroup region and hydrophobic lipid core. Through a number of downstream effects, this discrepancy can influence the way in which curvature modulates the behavior of membrane bound proteins depending on the adopted simulation model.

Cholesterol-rich lipid rafts are found to facilitate membrane fusion, central to processes like viral entry, fertilization, and neurotransmitter release. While the fusion process involves local, transient membrane dehydration, the impact of reduced hydration on cholesterol's structural organization in biological membranes remains unclear. Here, we employ confocal fluorescence microscopy and atomistic molecular dynamics simulations to investigate cholesterol behavior in phase-separated lipid bilayers under controlled hydration. We unveiled that dehydration prompts cholesterol release from raft-like domains into the surrounding fluid phase. Unsaturated phospholipids undergo more significant dehydration-induced structural changes and lose more hydrogen bonds with water than sphingomyelin. The results suggest that cholesterol redistribution is driven by the equalization of biophysical properties between phases and the need to satisfy lipid hydrogen bonds. This underscores the role of cholesterol-phospholipid-water interplay in governing cholesterol affinity for a specific lipid type, providing a new perspective on the regulatory role of cell membrane heterogeneity during membrane fusion.

Membrane fusion is a fundamental process that is exploited by enveloped viruses to enter host cells. In the case of the influenza virus, fusion is facilitated by the trimeric viral hemagglutinin protein (HA). So far, major focus has been put on its N-terminal fusion peptides, which are directly responsible for fusion initiation. A growing body of evidence points also to a significant functional role of the HA C-terminal domain, which however remains incompletely understood. Our computational study aimed to elucidate the structural and functional interdependencies within the HA C-terminal region encompassing the transmembrane domain (TMD) and the cytoplasmic tail (CT). In particular, we were interested in the conformational shift of the TMD in response to varying cholesterol concentration in the viral membrane and in its modulation by the presence of CT. Using free-energy calculations based on atomistic molecular dynamics simulations, we characterized transitions between straight and tilted metastable TMD configurations under varying conditions. We found that the presence of CT is essential for achieving a stable, highly tilted TMD configuration. As we demonstrate, such a configuration of HA membrane anchor likely supports the tilting motion of its ectodomain, which needs to be executed during membrane fusion. This finding highlights the functional role of, so far, the relatively overlooked CT region.

Membrane fusion is a fundamental process exploited by enveloped viruses to enter host cells. In the case of influenza virus, fusion is facilitated by the trimeric viral hemagglutinin protein (HA). So far, major focus has been put on its N-terminal fusion peptides, which are directly responsible for fusion initiation. A growing body of evidence points also to a significant functional role of HA C-terminal domain, which however remains incompletely understood. Our computational study aimed to elucidate the structural and functional interdependencies within the HA C-terminal region, encompassing the transmembrane domain (TMD) and the cytoplasmic tail (CT). In particular, we were interested in conformational shift of the TMD in response to varying cholesterol concentration in the viral membrane and in its modulation by the presence of CT. Using free energy calculations based on atomistic molecular dynamics simulations, we characterized transitions between straight and tilted metastable TMD configurations under varying conditions. We found that the presence of CT is essential for achieving a stable, highly tilted TMD configuration. As we demonstrate, such configuration of HA membrane anchor likely supports the tilting motion of its ectodomain which needs to be executed during membrane fusion. This finding highlights the functional role of, so far relatively overlooked, CT region.

The entry of influenza virus into the host cell requires fusion of its lipid envelope with the host membrane. It is catalysed by viral hemagglutinin protein, whose fragments called fusion peptides become inserted into the target bilayer and initiate its merging with the viral membrane. Isolated fusion peptides are already capable of inducing lipid mixing between liposomes. Years of studies indicate that upon membrane binding they form bend helical structure whose degree of opening fluctuates between tightly closed hairpin and an extended boomerang. The actual way in which they initiate fusion remains elusive. In this work we employ atomistic simulations of wild type and fusion inactive W14A mutant of influenza fusion peptides confined between two closely apposed lipid bilayers. We characterise peptide induced membrane perturbation and determine the potential of mean force for the formation of the first fusion intermediate, an interbilayer lipid bridge called stalk. Our results demonstrate two routes through which the peptides can lower free energy barrier towards fusion. The first one assumes peptides capability to adopt transmembrane configuration which subsequently promotes the creation of a stalk-hole complex. The second involves surface bound peptide configuration and proceeds owing to its ability to stabilise stalk by fitting into the region of extreme negative membrane curvature resulting from its formation. In both cases, the active peptide conformation corresponds to tight helical hairpin, whereas extended boomerang geometry appears to be unable to provide favourable thermodynamic effect. The latter observation offers plausible explanation for long known inactivity of boomerang-stabilising W14A mutation.

The entry of influenza virus into the host cell requires fusion of its lipid envelope with the host membrane. It is catalysed by viral hemagglutinin protein, whose fragments called fusion peptides become inserted into the target bilayer and initiate its merging with the viral membrane. Isolated fusion peptides are already capable of inducing lipid mixing between liposomes. Years of studies indicate that upon membrane binding they form bend helical structure whose degree of opening fluctuates between tightly closed hairpin and an extended boomerang. The actual way in which they initiate fusion remains elusive. In this work we employ atomistic simulations of wild type and fusion inactive W14A mutant of influenza fusion peptides confined between two closely apposed lipid bilayers. We characterise peptide induced membrane perturbation and determine the potential of mean force for the formation of the first fusion intermediate, an interbilayer lipid bridge called stalk. Our results demonstrate two routes through which the peptides can lower free energy barrier towards fusion. The first one assumes peptides capability to adopt transmembrane configuration which subsequently promotes the creation of a stalk-hole complex. The second involves surface bound peptide configuration and proceeds owing to its ability to stabilise stalk by fitting into the region of extreme negative membrane curvature resulting from its formation. In both cases, the active peptide conformation corresponds to tight helical hairpin, whereas extended boomerang geometry appears to be unable to provide favourable thermodynamic effect. The latter observation offers plausible explanation for long known inactivity of boomerang-stabilising W14A mutation.

Antimicrobial peptides emerge as compounds that can alleviate the global health hazard of antimicrobial resistance, prompting a need for novel computational approaches to peptide generation. Here, we propose HydrAMP, a conditional variational autoencoder that learns lower-dimensional, continuous representation of peptides and captures their antimicrobial properties. The model disentangles the learnt representation of a peptide from its antimicrobial conditions and leverages parameter-controlled creativity. HydrAMP is the first model that is directly optimized for diverse tasks, including unconstrained and analogue generation and outperforms other approaches in these tasks. An additional preselection procedure based on ranking of generated peptides and molecular dynamics simulations increases experimental validation rate. Wet-lab experiments on five bacterial strains confirm high activity of nine peptides generated as analogues of clinically relevant prototypes, as well as six analogues of an inactive peptide. HydrAMP enables generation of diverse and potent peptides, making a step towards resolving the antimicrobial resistance crisis.

Cannabinoid receptor type 1 (CB1) is an important modulator of many key physiological functions and thus a compelling molecular target. However, safe CB1 targeting is a non-trivial task. In recent years, there has been a surge of data indicating that drugs successfully used in the clinic for years (e.g. paracetamol) show CB1 activity. Moreover, there is a lot of promise in finding CB1 ligands in plants other than Cannabis sativa. In this study, we searched for possible CB1 activity among already existing drugs, their metabolites, phytochemicals, and natural-like molecules. We conducted two iterations of virtual screening, verifying the results with in vitro binding and functional assays. The in silico procedure consisted of a wide range of structure- and ligand-based methods, including docking, molecular dynamics, and quantitative structure-activity relationship (QSAR). As a result, we identified travoprost and ginkgetin as CB1 ligands, which provides a starting point for future research on the impact of their metabolites or preparations on the endocannabinoid system. Moreover, we found five natural-like compounds with submicromolar or low micromolar affinity to CB1, including one mixed partial agonist/antagonist viable for hit-to-lead phase. Finally, the computational procedure established in this work will be of use for future screening campaigns for novel CB1 ligands.