Dehydration of Lipid Membranes Drives Redistribution of Cholesterol Between Lateral Domains.

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.

Molecular Mechanisms behind Conformational Transitions of the Influenza Virus Hemagglutinin Membrane Anchor.

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.

Two modes of fusogenic action for influenza virus fusion peptide.

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.

Discovering highly potent antimicrobial peptides with deep generative model HydrAMP.

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.

Identification of CB1 Ligands among Drugs, Phytochemicals and Natural-Like Compounds: Virtual Screening and In Vitro Verification.

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.

Membrane-Bound Configuration and Lipid Perturbing Effects of Hemagglutinin Subunit 2 N-Terminus Investigated by Computer Simulations.

Hemagglutinin (HA) mediated fusion of influenza virus envelope with host lipid membrane is a critical step warrantying virus entry to the cell. Despite tremendous advances in structural biology methods, the knowledge concerning the details of HA2 subunit insertion into the target membrane and its subsequent bilayer perturbing effect is still rather limited. Herein, based on a set of molecular dynamics simulations, we investigate the structure and interaction with lipid membrane of the N-terminal HA2 region comprising a trimer of fusion peptides (HAfps) tethered by flexible linkers to a fragment of coiled-coil stem structure. We find that, prior to insertion into the membrane, HAfps within the trimers do not sample space individually but rather associate into a compact hydrophobic aggregate. Once within the membrane, they fold into tight helical hairpins, which remain at the lipid-water interface. However, they can also assume stable, membrane-spanning configurations of significantly increased membrane-perturbing potential. In this latter case, HAfps trimers centre around the well-hydrated transmembrane channel-forming distinct, symmetric assemblies, whose wedge-like shape may play a role in promoting membrane curvature. We also demonstrate that, following HAfps insertion, the coiled-coil stem spontaneously tilts to almost membrane-parallel orientation, reflecting experimentally observed configuration adopted in the course of membrane fusion by complete HA2 units at the rim of membrane contact zones.

Granger Causality Analysis of Chignolin Folding.

Constantly advancing computer simulations of biomolecules provide huge amounts of data that are difficult to interpret. In particular, obtaining insights into functional aspects of macromolecular dynamics, often related to cascades of transient events, calls for methodologies that depart from the well-grounded framework of equilibrium statistical physics. One of the approaches toward the analysis of complex temporal data which has found applications in the fields of neuroscience and econometrics is Granger causality analysis. It allows determining which components of multidimensional time series are most influential for the evolution of the entire system, thus providing insights into causal relations within the dynamic structure of interest. In this work, we apply Granger analysis to a long molecular dynamics trajectory depicting repetitive folding and unfolding of a mini ß-hairpin protein, CLN025. We find objective, quantitative evidence indicating that rearrangements within the hairpin turn region are determinant for protein folding and unfolding. On the contrary, interactions between hairpin arms score low on the causality scale. Taken together, these findings clearly favor the concept of zipperlike folding, which is one of two postulated ß-hairpin folding mechanisms. More importantly, the results demonstrate the possibility of a conclusive application of Granger causality analysis to a biomolecular system.

Transient Excursions to Membrane Core as Determinants of Influenza Virus Fusion Peptide Activity.

Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and initiate lipid exchange. These isolated fragments, known as fusion peptides (HAfp), already possess own fusogenic activity towards liposomes. Although they have long been studied with the hope to uncover the details of HA-mediated fusion, their actual mechanism of action remains elusive. Here, we use extensive molecular dynamics simulations combined with experimental studies of three HAfp variants to fully characterize their free energy landscape and interaction with lipid bilayer. In addition to customary assumed peptides localization at lipid-water interface, we characterize membrane-spanning configurations, which turn out to be metastable for active HAfps and unstable for the fusion inactive W14A mutant. We show that, while the degree of membrane perturbation by surface peptide configurations is relatively low and does not show any mutation-related differences, the effect of deeply inserted configurations is significant and correlates with insertion depth of the N-terminal amino group which is the highest for the wild type HAfp. Finally, we demonstrate the feasibility of spontaneous peptide transition to intramembrane location and the critical role of strictly conserved tryptofan residue 14 in this process.

Entropy-based distance cutoff for protein internal contact networks.

Protein structure networks (PSNs) have long been used to provide a coarse yet meaningful representation of protein structure, dynamics, and internal communication pathways. An important question is what criteria should be applied to construct the network so that to include relevant interresidue contacts while avoiding unnecessary connections. To address this issue, we systematically considered varying residue distance cutoff length and the probability threshold for contact formation to construct PSNs based on atomistic molecular dynamics in order to assess the amount of mutual information within the resulting representations. We found that the minimum in mutual information is universally achieved at the cutoff length of 5 Å, irrespective of the applied contact formation probability threshold in all considered, distinct proteins. Assuming that the optimal PSNs should be characterized by the least amount of redundancy, which corresponds to the minimum in mutual information, this finding suggests an objective criterion for cutoff distance and supports the existing preference towards its customary selection around 5 Å length, typically based to date on heuristic criteria.

GridSolvate: A Web Server for the Prediction of Biomolecular Hydration Properties.

We present a novel web server, named gridSolvate, dedicated to the prediction of biomolecular hydration properties. Given a solute in atomic representation, such as a protein or protein-ligand complex, the server determines positions and excess chemical potential of buried and first hydration shell water molecules. Calculations are based on our semiexplicit hydration model that provides computational efficiency close to implicit solvent approaches, yet captures a number of physical effects unique to explicit solvent representation. The model was introduced and validated before in the context of bulk hydration of drug-like solutes and determination of protein hydration sites. Current methodological developments merge those two avenues into a single, easily accessible tool. Here, we focus on the server's ability to predict water distribution and affinity within protein-ligand interfaces. We demonstrate that with possibly minimal user intervention the server correctly predicts the locations of 77% of interface water molecules in an external set of test structures. The server is freely available at https://gsolvate.biomod.cent.uw.edu.pl.

Conserved internal hydration motifs in protein kinases.

Crystal structures of diverse protein kinase catalytic subunits reveal a number of water molecules whose positions within the protein core are better preserved than amino acid types in many functionally important locations. It remains unknown whether they play any particular role, and whether their removal, disturbing local interaction patterns to no smaller degree than amino acid mutations, can affect kinase stability and function. In this study, we apply an array of computational approaches to characterize hydration of kinase catalytic subunits. First, we systematically screen multiple crystal structures with the use of a simplified hydration model in order to determine the distribution of internal solvent and the degree of its conservation. Second, we analyze water structure, dynamics and binding affinity to buried hydration sites in a number of kinases, also taking into account their variable functional state. We find that a large portion of buried solvent is dynamic, possibly contributing to kinase conformational changes related to the activation process. In turn, binding free energies of some of tightly bound conserved water molecules to different kinases tend to shift in a similar manner following the change of their functional state. This finding highlights the likely specific role of internal solvent in fine tuning local protein plasticity.

Restriction of S-adenosylmethionine conformational freedom by knotted protein binding sites.

S-adenosylmethionine (SAM) is one of the most important enzyme substrates. It is vital for the function of various proteins, including large group of methyltransferases (MTs). Intriguingly, some bacterial and eukaryotic MTs, while catalysing the same reaction, possess significantly different topologies, with the former being a knotted one. Here, we conducted a comprehensive analysis of SAM conformational space and factors that affect its vastness. We investigated SAM in two forms: free in water (via NMR studies and explicit solvent simulations) and bound to proteins (based on all data available in the PDB and on all-atom molecular dynamics simulations in water). We identified structural descriptors-angles which show the major differences in SAM conformation between unknotted and knotted methyltransferases. Moreover, we report that this is caused mainly by a characteristic for knotted MTs compact binding site formed by the knot and the presence of adenine-binding loop. Additionally, we elucidate conformational restrictions imposed on SAM molecules by other protein groups in comparison to conformational space in water.

Quick temperature-sweep pure-shift NMR: the case of solvent effects in atorvastatin.

Pure-shift NMR experiments provide highly resolved spectra, which could be perfect for precise monitoring of chemical shift variations under different conditions, such as temperature or concentration. However, their sensitivity is relatively low and signal sampling is time-consuming, which leads to long experimental times, making such serial acquisition problematic. In this paper we present a new method of NMR spectroscopy which improves the speed and sensitivity of serial pseudo-two-dimensional pure-shift experiments. The example of variable-temperature study of atorvastatin reveals the potential of the method in verifying the theoretical predictions of solvent-dependent spectral effects.

Affinity, kinetics, and pathways of anisotropic ligands binding to hydrophobic model pockets.

Using explicit-water molecular dynamics simulations of a generic pocket-ligand model, we investigate how chemical and shape anisotropy of small ligands influences the affinities, kinetic rates, and pathways for their association with hydrophobic binding sites. In particular, we investigate aromatic compounds, all of similar molecular size, but distinct by various hydrophilic or hydrophobic residues. We demonstrate that the most hydrophobic sections are in general desolvated primarily upon binding to the cavity, suggesting that specific hydration of the different chemical units can steer the orientation pathways via a "hydrophobic torque." Moreover, we find that ligands with bimodal orientation fluctuations have significantly increased kinetic barriers for binding compared to the kinetic barriers previously observed for spherical ligands due to translational fluctuations. We exemplify that these kinetic barriers, which are ligand specific, impact both binding and unbinding times for which we observe considerable differences between our studied ligands.

Water-mediated conformational preselection mechanism in substrate binding cooperativity to protein kinase A.

Substrate binding cooperativity in protein kinase A (PKA) seems to involve allosteric coupling between the two binding sites. It received significant attention, but its molecular basis still remains not entirely clear. Based on long molecular dynamics of PKA and its complexes, we characterized an allosteric pathway that links ATP binding to the redistribution of states adopted by a protein substrate positioning segment in favor of those that warrant correct binding. We demonstrate that the cooperativity mechanism critically depends on the presence of water in two distinct, buried hydration sites. One holds just a single water molecule, which acts as a switchable hydrogen bond bridge along the allosteric pathway. The second, filled with partially disordered solvent, is essential for providing a smooth free energy landscape underlying conformational transitions of the peptide binding region. Our findings remain in agreement with experimental data, also concerning the cooperativity abolishing effect of the Y204A mutation, and indicate a plausible molecular mechanism contributing to experimentally observed binding cooperativity of the two substrates.

Charged N-terminus of Influenza Fusion Peptide Facilitates Membrane Fusion.

Cleavage of hemagglutinin precursor (HA0) by cellular proteases results in the formation of two subunits, HA1 and HA2. The N-terminal fragment of HA2, named a fusion peptide (HAfp), possess a charged, amine N-terminus. It has been shown that the N-terminus of HAfp stabilizes the structure of a helical hairpin observed for a 23-amino acid long peptide (HAfp1-23), whose larger activity than HAfp1-20 has been demonstrated recently. In this paper, we analyze the effect of N-terminal charge on peptide-mediated fusion efficiency and conformation changes at the membrane interface by comparison with the corresponding N-acetylated peptides of 20- and 23-amino acid lengths. We found that higher fusogenic activities of peptides with unmodified amino termini correlates with their ability to form helical hairpin structures oriented perpendicularly to the membrane plane. Molecular dynamics simulations showed that acetylated peptides adopt open and surface-bound conformation more often, which induced less disorder of the phospholipid chains, as compared to species with unmodified amino termini.

Principles for Tuning Hydrophobic Ligand-Receptor Binding Kinetics.

We investigate how to tune the rate of hydrophobic ligand-receptor association due to the role of solvent in adjustable receptor pockets by explicit-water molecular dynamics (MD) simulations. Our model considers the binding of a spherical ligand (key/guest) to a concave surface recess in a nonpolar wall as receptor (lock/host). We systematically modify the receptor's physicochemical properties in terms of geometry and dispersion attraction which, in turn, alter the water occupancy and fluctuations within the pocket. We demonstrate that even minor pocket modifications can lead to a significant acceleration of the water-mediated association. For example, the binding switches from comparably slow to fast if the binding pocket becomes only slightly deeper. We find that the degree of hydrophobicity, characterized by hydration occupancy and its fluctuations, clearly correlates with the binding times and, for instance, links the sudden acceleration to an abrupt increase in hydrophobicity. For a deeper analysis based on passage time theory, we quantify the intimate coupling between solvent fluctuations and the ligand's local dynamics and friction. The coupling exhibits substantial nonequilibrium effects and maximizes shortly before binding, which slows down the binding kinetics in all cases. In summary, we rationalize how the physicochemical properties of a nonpolar, concave binding site tune key-lock binding kinetics due to water-mediated forces and fluctuations. Our study thus complements the profound understanding of the solvent's influence in host-guest binding, which is essential for tailored solutions in catalysis and pharmaceutical applications.

Explicit Solvent Hydration Benchmark for Proteins with Application to the PBSA Method.

Explicit and implicit solvent models have a proven record of delivering hydration free energies of small, druglike solutes in reasonable agreement with experiment. Hydration of macromolecules, such as proteins, is to a large extent uncharted territory, with few results shedding light on quantitative consistency between different solvent models, let alone their ability to reproduce real water. In this work, based on extensive explicit solvent simulations employing TIP3P and SPC/E water models we analyze hydration free energy changes between fixed conformations of 5 diverse proteins, including large multidomain structures. For the two solvent models we find better agreement in electrostatic rather than nonpolar contributions (RMSE of 2.3 and 2.7 kcal/mol, respectively), even though absolute values of the latter are typically an order of magnitude smaller. We also highlight the importance of finite size corrections to relative protein hydration free energies, which turn out to be rather large, on the order of several kcal/mol, and are necessary for proper interpretation of results obtained under periodic boundary conditions. We further compare gathered data with predictions of the implicit solvent approach based on the Poisson equation and the surface or volume based nonpolar term. We find definitely lesser consistency than between the two explicit models (RMSE between implicit and TIP3 results of 11.3 and 8.4 kcal/mol for electrostatic and nonpolar contributions, respectively). In the process we determine the value of the protein dielectric constant and the geometric model for the dielectric boundary that provide for the best agreement. Finally, we evaluate the usefulness of surface and volume based models of nonpolar contributions to hydration free energy of large biomolecules.

Three conserved C-terminal residues of influenza fusion peptide alter its behavior at the membrane interface.

The N-terminal fragment of the viral hemagglutinin HA2 subunit is termed a fusion peptide (HAfp). The 23-amino acid peptide (HAfp1-23) contains three C-terminal W21-Y22-G23 residues which are highly conserved among serotypes of influenza A and has been shown to form a tight helical hairpin very distinct from the boomerang structure of HAfp1-20. We studied the effect of peptide length on fusion properties, structural dynamics, and binding to the membrane interface. We developed a novel fusion visualization assay based on FLIM microscopy on giant unilamellar vesicles (GUV). By means of molecular dynamics simulations and spectroscopic measurements, we show that the presence of the three C-terminal W21-Y22-G23 residues promotes the hairpin formation, which orients perpendicularly to the membrane plane and induces more disorder in the surrounding lipids than the less structured HAfp1-20. Moreover, we report cholesterol-enriched domain formation induced exclusively by the longer fusion peptide.