Optimizing properties of translocation-enhancing transmembrane proteins.

Cell membranes act as semi-permeable barriers, often restricting the entry of large or hydrophilic molecules. Nonetheless, certain amphiphilic molecules, such as antimicrobial and cell-penetrating peptides, can cross these barriers. In this study, we demonstrate that specific properties of transmembrane proteins/peptides can enhance membrane permeation of amphiphilic peptides. Using coarse-grained molecular dynamics with free-energy calculations, we identify key translocation-enhancing attributes of transmembrane proteins/peptides: a continuous hydrophilic patch, charged residues preferably in the membrane center, and aromatic hydrophobic residues. By employing both coarse-grained and atomistic simulations, complemented by experimental validation, we show that these properties not only enhance peptide translocation but also speed up lipid flip-flop. The enhanced flip-flop reinforces the idea that proteins such as scramblases and insertases not only share structural features but also operate through identical biophysical mechanisms enhancing the insertion and translocation of amphiphilic molecules. Our insights offer guidelines for the designing of translocation-enhancing proteins/peptides that could be used in medical and biotechnological applications.

Nanoparticle induced fusion of lipid membranes.

Membrane fusion is crucial for infection of enveloped viruses, cellular transport, and drug delivery via liposomes. Nanoparticles can serve as fusogenic agents facilitating such membrane fusion for direct transmembrane transport. However, the underlying mechanisms of nanoparticle-induced fusion and the ideal properties of such nanoparticles remain largely unknown. Here, we used molecular dynamics simulations to investigate the efficacy of spheroidal nanoparticles with different size, prolateness, and ligand interaction strengths to enhance fusion between vesicles. By systematically varying nanoparticle properties, we identified how each parameter affects the fusion process and determined the optimal parameter range that promotes fusion. These findings provide valuable insights for the design and optimization of fusogenic nanoparticles with potential biotechnological and biomedical applications.

Valency of Ligand-Receptor Binding from Pair Potentials.

Coarse grained molecular dynamics simulations have been crucial for investigating the dynamics of nanoparticle uptake by cell membranes via ligand-receptor interactions. These models have enabled researchers to evaluate the effects of nanoparticle size, shape, and ligand distribution on cellular uptake. However, when pair potentials are used to represent ligand-receptor interactions, the number of receptors interacting with one ligand, valency, may vary. We demonstrate that the curvature of a nanoparticle, strength of ligand-receptor interactions, and ligand or receptor concentration change the valency, ranging from 3.4 to 5.1 in this study. Such a change in valency can create inaccurate comparisons between nanoparticles or even result in the uptake of smaller nanoparticles than would be expected. To rectify this inconsistency, we propose the adoption of a model based on bond formation and use it to determine the extent to which previous studies may have been affected. This work recommends avoiding pair potentials for modeling ligand-receptor interactions to ensure methodological consistency in nanoparticle studies.

Peptide translocation across asymmetric phospholipid membranes.

The transport of molecules across cell membranes is vital for proper cell function and effective drug delivery. While most cell membranes naturally possess an asymmetric lipid composition, research on membrane transport predominantly uses symmetric lipid membranes. The permeation through the asymmetric membrane is then calculated as a sum of the inverse permeabilities of leaflets from symmetric bilayers. In this study, we examined how two types of amphiphilic molecules translocate across both asymmetric and symmetric membranes. Using computer simulations with both coarse-grained and atomistic force fields, we calculated the free energy profiles for the passage of model amphiphilic peptides and a lipid across various membranes. Our results consistently demonstrate that while the free energy profiles for asymmetric membranes with a small differential stress concur with symmetric ones in the region of lipid headgroups, the profiles differ around the center of the membrane. In this region, the free energy for the asymmetric membrane transitions between the profiles for two symmetric membranes. In addition, we show that peptide permeability through an asymmetric membrane cannot always be predicted from the permeabilities of the symmetric membranes. This indicates that using symmetric membranes falls short in providing an accurate depiction of peptide translocation across asymmetric membranes.

Insertases scramble lipids: Molecular simulations of MTCH2.

Scramblases play a pivotal role in facilitating bidirectional lipid transport across cell membranes, thereby influencing lipid metabolism, membrane homeostasis, and cellular signaling. MTCH2, a mitochondrial outer membrane protein insertase, has a membrane-spanning hydrophilic groove resembling those that form the lipid transit pathway in known scramblases. Employing both coarse-grained and atomistic molecular dynamics simulations, we show that MTCH2 significantly reduces the free energy barrier for lipid movement along the groove and therefore can indeed function as a scramblase. Notably, the scrambling rate of MTCH2 in silico is similar to that of voltage-dependent anion channel (VDAC), a recently discovered scramblase of the outer mitochondrial membrane, suggesting a potential complementary physiological role for these mitochondrial proteins. Finally, our findings suggest that other insertases which possess a hydrophilic path across the membrane like MTCH2, can also function as scramblases.

Amphipathic Helices Can Sense Both Positive and Negative Curvatures of Lipid Membranes.

Curvature sensing is an essential ability of biomolecules to preferentially localize to membrane regions of a specific curvature. It has been shown that amphipathic helices (AHs), helical peptides with both hydrophilic and hydrophobic regions, could sense a positive membrane curvature. The origin of this AH sensing has been attributed to their ability to exploit lipid-packing defects that are enhanced in regions of positive curvature. In this study, we revisit an alternative framework where AHs act as sensors of local internal stress within the membrane, suggesting the possibility of an AH sensing a negative membrane curvature. Using molecular dynamics simulations, we gradually tuned the hydrophobicity of AHs, thereby adjusting their insertion depth so that the curvature preference of AHs is switched from positive to negative. This study suggests that highly hydrophobic AHs could preferentially localize proteins to regions of a negative membrane curvature.

Phospholipids are imported into mitochondria by VDAC, a dimeric beta barrel scramblase.

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identify the Voltage Dependent Anion Channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act to import lipids into mitochondria.

Insertases Scramble Lipids: Molecular Simulations of MTCH2.

Scramblases play a pivotal role in facilitating bidirectional lipid transport across cell membranes, thereby influencing lipid metabolism, membrane homeostasis, and cellular signaling. MTCH2, a mitochondrial outer membrane protein insertase, has a membrane-spanning hydrophilic groove resembling those that form the lipid transit pathway in known scramblases. Employing both coarse-grained and atomistic molecular dynamics simulations, we show that MTCH2 significantly reduces the free energy barrier for lipid movement along the groove and therefore can indeed function as a scramblase. Notably, the scrambling rate of MTCH2 in silico is similar to that of VDAC, a recently discovered scramblase of the outer mitochondrial membrane, suggesting a potential complementary physiological role for these mitochondrial proteins. Finally, our findings suggest that other insertases which possess a hydrophilic path across the membrane like MTCH2, can also function as scramblases.

Magainin 2 and PGLa in bacterial membrane mimics IV: Membrane curvature and partitioning.

We previously reported that the synergistically enhanced antimicrobial activity of magainin 2 (MG2a) and PGLa is related to membrane adhesion and fusion. Here, we demonstrate that equimolar mixtures of MG2a and L18W-PGLa induce positive monolayer curvature stress and sense, at the same time, positive mean and Gaussian bilayer curvatures already at low amounts of bound peptide. The combination of both abilities-membrane curvature sensing and inducing-is most likely the base for the synergistically enhanced peptide activity. In addition, our coarse-grained simulations suggest that fusion stalks are promoted by decreasing the free-energy barrier for their formation rather than by stabilizing their shape. We also interrogated peptide partitioning as a function of lipid and peptide concentration using tryptophan fluorescence spectroscopy and peptide-induced leakage of dyes from lipid vesicles. In agreement with a previous report, we find increased membrane partitioning of L18W-PGLa in the presence of MG2a. However, this effect does not prevail to lipid concentrations higher than 1 mM, above which all peptides associate with the lipid bilayers. This implies that synergistic effects of MG2a and L18W-PGLa in previously reported experiments with lipid concentrations >1 mM are due to peptide-induced membrane remodeling and not their specific membrane partitioning.

Binding of DEP domain to phospholipid membranes: More than just electrostatics.

Over the past decades an extensive effort has been made to provide a more comprehensive understanding of Wnt signaling, yet many regulatory and structural aspects remain elusive. Among these, the ability of Dishevelled (DVL) protein to relocalize at the plasma membrane is a crucial step in the activation of all Wnt pathways. The membrane binding of DVL was suggested to be mediated by the preferential interaction of its C-terminal DEP domain with phosphatidic acid (PA). However, due to the scarcity and fast turnover of PA, we investigated the role on the membrane association of other more abundant phospholipids. The combined results from computational simulations and experimental measurements with various model phospholipid membranes, demonstrate that the membrane binding of DEP/DVL constructs is governed by the concerted action of generic electrostatics and finely-tuned intermolecular interactions with individual lipid species. In particular, while we confirmed the strong preference for PA lipid, we also observed a weak but non-negligible affinity for phosphatidylserine, the most abundant anionic phospholipid in the plasma membrane, and phosphatidylinositol 4,5-bisphosphate. The obtained molecular insight into DEP-membrane interaction helps to elucidate the relation between changes in the local membrane composition and the spatiotemporal localization of DVL and, possibly, other DEP-containing proteins.

Synthesis and Profiling of Highly Selective Inhibitors of Methyltransferase DOT1L Based on Carbocyclic C-Nucleosides.

Histone methyltransferase DOT1L is an attractive therapeutic target for the treatment of hematological malignancies. Here, we report the design, synthesis, and profiling of new DOT1L inhibitors based on nonroutine carbocyclic C-nucleoside scaffolds. The experimentally observed SAR was found to be nontrivial as seemingly minor changes of individual substituents resulted in significant changes in the affinity to DOT1L. Molecular modeling suggested that these trends could be related to significant conformational changes of the protein upon interaction with the inhibitors. The compounds 22 and (-)-53 (MU1656), carbocyclic C-nucleoside analogues of the natural nucleoside derivative EPZ004777, and the clinical candidate EPZ5676 (pinometostat) potently and selectively inhibit DOT1L in vitro as well as in the cell. The most potent compound MU1656 was found to be more metabolically stable and significantly less toxic in vivo than pinometostat itself.

Magainin 2 and PGLa in bacterial membrane mimics III: Membrane fusion and disruption.

We previously speculated that the synergistically enhanced antimicrobial activity of Magainin 2 and PGLa is related to membrane adhesion, fusion, and further membrane remodeling. Here we combined computer simulations with time-resolved in vitro fluorescence microscopy, cryoelectron microscopy, and small-angle X-ray scattering to interrogate such morphological and topological changes of vesicles at nanoscopic and microscopic length scales in real time. Coarse-grained simulations revealed formation of an elongated and bent fusion zone between vesicles in the presence of equimolar peptide mixtures. Vesicle adhesion and fusion were observed to occur within a few seconds by cryoelectron microscopy and corroborated by small-angle X-ray scattering measurements. The latter experiments indicated continued and time-extended structural remodeling for individual peptides or chemically linked peptide heterodimers but with different kinetics. Fluorescence microscopy further captured peptide-dependent adhesion, fusion, and occasional bursting of giant unilamellar vesicles a few seconds after peptide addition. The synergistic interactions between the peptides shorten the time response of vesicles and enhance membrane fusogenic and disruption properties of the equimolar mixture compared with the individual peptides.

Cargo Release from Nonenveloped Viruses and Virus-like Nanoparticles: Capsid Rupture or Pore Formation.

Virus-like nanoparticles are protein shells similar to wild-type viruses, and both aim to deliver their content into a cell. Unfortunately, the release mechanism of their cargo/genome remains elusive. Pores on the symmetry axes were proposed to enable the slow release of the viral genome. In contrast, cryo-EM images showed that capsids of nonenveloped RNA viruses can crack open and rapidly release the genome. We combined in vitro cryo-EM observations of the genome release of three viruses with coarse-grained simulations of generic virus-like nanoparticles to investigate the cargo/genome release pathways. Simulations provided details on both slow and rapid release pathways, including the success rates of individual releases. Moreover, the simulated structures from the rapid release pathway were in agreement with the experiment. Slow release occurred when interactions between capsid subunits were long-ranged, and the cargo/genome was noncompact. In contrast, rapid release was preferred when the interaction range was short and/or the cargo/genome was compact. These findings indicate a design strategy of virus-like nanoparticles for drug delivery.

The impact of the glycan headgroup on the nanoscopic segregation of gangliosides.

Gangliosides form an important class of receptor lipids containing a large oligosaccharide headgroup whose ability to self-organize within lipid membranes results in the formation of nanoscopic platforms. Despite their biological importance, the molecular basis for the nanoscopic segregation of gangliosides is not clear. In this work, we investigated the role of the ganglioside headgroup on the nanoscale organization of gangliosides. We studied the effect of the reduction in the number of sugar units of the ganglioside oligosaccharide chain on the ability of gangliosides GM1, GM2, and GM3 to spontaneously self-organize into lipid nanodomains. To reach nanoscopic resolution and to identify molecular forces that drive ganglioside segregation, we combined an experimental technique, Förster resonance energy transfer analyzed by Monte-Carlo simulations offering high lateral and trans-bilayer resolution with molecular dynamics simulations. We show that the ganglioside headgroup plays a key role in ganglioside self-assembly despite the negative charge of the sialic acid group. The nanodomains range from 7 to 120 nm in radius and are mostly composed of the surrounding bulk lipids, with gangliosides being a minor component of the nanodomains. The interactions between gangliosides are dominated by the hydrogen bonding network between the headgroups, which facilitates ganglioside clustering. The N-acetylgalactosamine sugar moiety of GM2, however, seems to impair the stability of these clusters by disrupting hydrogen bonding of neighboring sugars, which is in agreement with a broad size distribution of GM2 nanodomains. The simulations suggest that the formation of nanodomains is likely accompanied by several conformational changes in the gangliosides, which, however, have little impact on the solvent exposure of these receptor groups. Overall, this work identifies the key physicochemical factors that drive nanoscopic segregation of gangliosides.

Cardiolipin-Containing Lipid Membranes Attract the Bacterial Cell Division Protein DivIVA.

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane.

Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins.

Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.

Advances in Molecular Understanding of α-Helical Membrane-Active Peptides.

Biological membranes separate the interior of cells or cellular compartments from their outer environments. This barrier function of membranes can be disrupted by membrane-active peptides, some of which can spontaneously penetrate through the membranes or open leaky transmembrane pores. However, the origin of their activity/toxicity is not sufficiently understood for the development of more potent peptides. To this day, there are no design rules that would be generally valid, and the role of individual amino acids tends to be sequence-specific.In this Account, we describe recent progress in understanding the design principles that govern the activity of membrane-active peptides. We focus on α-helical amphiphilic peptides and their ability to (1) translocate across phospholipid bilayers, (2) form transmembrane pores, or (3) act synergistically, i.e., to produce a significantly more potent effect in a mixture than the individual components.We refined the description of peptide translocation using computer simulations and demonstrated the effect of selected residues. Our simulations showed the necessity to explicitly include charged residues in the translocation description to correctly sample the membrane perturbations they can cause. Using this description, we calculated the translocation of helical peptides with and without the kink induced by the proline/glycine residue. The presence of the kink had no effect on the translocation barrier, but it decreased the peptide affinity to the membrane and reduced the peptide stability inside the membrane. Interestingly, the effects were mainly caused by the peptide's increased polarity, not the higher flexibility of the kink.Flexibility plays a crucial role in pore formation and affects distinct pore structures in different ways. The presence of a kink destabilizes barrel-stave pores, because the kink prevents the tight packing of peptides in the bundle, which is characteristic of the barrel-stave structure. In contrast, the kink facilitates the formation of toroidal pores, where the peptides are only loosely arranged and do not need to closely assemble. The exact position of the kink in the sequence further determines the preferred arrangement of peptides in the pore, i.e., an hourglass or U-shaped structure. In addition, we demonstrated that two self-associated (via termini) helical peptides could mimic the behavior of peptides with a helix-kink-helix motif.Finally, we review the recent findings on the peptide synergism of the archetypal mixture of Magainin 2 and PGLa peptides. We focused on a bacterial plasma membrane mimic that contains negatively charged lipids and lipids with negative intrinsic curvature. We showed that the synergistic action of peptides was highly dependent on the lipid composition. When the lipid composition and peptide/lipid ratios were changed, the systems exhibited more complex behavior than just the previously reported pore formation. We observed membrane adhesion, fusion, and even the formation of the sponge phase in this regime. Furthermore, enhanced adhesion/partitioning to the membrane was reported to be caused by lipid-induced peptide aggregation.In conclusion, the provided molecular insight into the complex behavior of membrane-active peptides provides clues for the design and modification of antimicrobial peptides or toxins.

Capsid opening enables genome release of iflaviruses.

The family Iflaviridae includes economically important viruses of the western honeybee such as deformed wing virus, slow bee paralysis virus, and sacbrood virus. Iflaviruses have nonenveloped virions and capsids organized with icosahedral symmetry. The genome release of iflaviruses can be induced in vitro by exposure to acidic pH, implying that they enter cells by endocytosis. Genome release intermediates of iflaviruses have not been structurally characterized. Here, we show that conformational changes and expansion of iflavirus RNA genomes, which are induced by acidic pH, trigger the opening of iflavirus particles. Capsids of slow bee paralysis virus and sacbrood virus crack into pieces. In contrast, capsids of deformed wing virus are more flexible and open like flowers to release their genomes. The large openings in iflavirus particles enable the fast exit of genomes from capsids, which decreases the probability of genome degradation by the RNases present in endosomes.