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Cholesterol plays a crucial role in biomembranes by regulating various properties such as fluidity, rigidity, permeability, and organization of lipid bilayers. The latest version of the Martini model, Martini 3, offers significant improvements in interaction balance, molecular packing, and the inclusion of new bead types and sizes. However, the release of the new model resulted in the need to re-parameterize many core molecules, including cholesterol. Here, we describe the development and validation of a Martini 3 cholesterol model, addressing issues related to its bonded setup, shape, volume and hydrophobicity. The proposed model mitigates some limitations of its Martini 2 predecessor while maintaining or improving overall behavior.

Phosphoinositides are a family of membrane phospholipids that play crucial roles in membrane regulatory events. As such, these lipids are often a key part of molecular dynamics simulation studies of biological membranes, in particular of those employing coarse-grain models because of the potential long times and sizes of the involved membrane processes. Version 3 of the widely used Martini coarse-grain force field has been recently published, greatly refining many aspects of biomolecular interactions. In order to properly use it for lipid membrane simulations with phosphoinositides, we put forth the Martini 3-specific parameterization of inositol, phosphatidylinositol, and seven physiologically relevant phosphorylated derivatives of phosphatidylinositol. Compared to parameterizations for earlier Martini versions, focus was put on a more accurate reproduction of the behavior seen in both atomistic simulations and experimental studies, including the signaling-relevant phosphoinositide interaction with divalent cations. The models that we develop improve upon the conformational dynamics of phosphoinositides in the Martini force field and provide stable topologies at typical Martini time steps. They are able to reproduce experimentally known protein-binding poses as well as phosphoinositide aggregation tendencies. The latter was tested both in the presence and absence of calcium and included correct behavior of PI(4,5)P

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed over 5 million people and is causing a devastating social and economic impact all over the world. The rise of new variants of concern (VOCs) represents a difficult challenge due to the loss vaccine and natural immunity, and increased transmissibility. All circulating VOCs contain mutations in the spike glycoprotein, which mediates fusion between the viral and host cell membranes, via its receptor binding domain (RBD) that binds to angiotensin-converting enzyme 2 (ACE2). In an attempt to understand the effect of RBD mutations in circulating VOCs, a lot of attention has been given to the RBD-ACE2 interaction. However, this type of analysis is limited, since it ignores more indirect effects, such as the conformational dynamics of the RBD itself. Observing that some VOCs mutations occur in residues that are not in direct contact with ACE2, we hypothesized that they could affect RBD conformational dynamics. To test this, we performed long atomistic (AA) molecular dynamics (MD) simulations to investigate the structural dynamics of wt RBD, and that of three circulating VOCs (alpha, beta, and delta). Our results show that in solution, wt RBD presents two distinct conformations: an “open” conformation where it is free to bind ACE2; and a “closed” conformation, where the RBM ridge blocks the binding surface. The alpha and beta variants significantly impact the open/closed equilibrium, shifting it towards the open conformation by roughly 20%. This shift likely increases ACE2 binding affinity. Simulations of the currently predominant delta variant RBD were extreme in this regard, in that a closed conformation was never observed. Instead, the system alternated between the before mentioned open conformation and an alternative “reversed” one, with a significantly changed orientation of the RBM ridge flanking the RBD. This alternate conformation could potentially provide a fitness advantage not only due to increased availability for ACE2 binding, but also by aiding antibody escape through epitope occlusion. These results support the hypothesis that VOCs, and particularly the delta variant, impact RBD conformational dynamics in a direction that simultaneously promotes efficient binding to ACE2 and antibody escape.

Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) plays a critical role in the regulation of various plasma membrane processes and signaling pathways in eukaryotes. A significant amount of cellular resources are spent on maintaining the dominant 1-stearoyl-2-arachidonyl PI(4,5)P2 acyl-chain composition, while less abundant and more saturated species become more prevalent in response to specific stimuli, stress or aging. Here, we report the impact of acyl-chain structure on the biophysical properties of cation-induced PI(4,5)P2 nanodomains. PI(4,5)P2 species with increasing levels of acyl-chain saturation cluster in progressively more ordered nanodomains, culminating in the formation of gel-like nanodomains for fully saturated species. The formation of these gel-like domains was largely abrogated in the presence of 1-stearoyl-2-arachidonyl PI(4,5)P2. This is, to the best of our knowledge, the first report of the impact of PI(4,5)P2 acyl-chain composition on cation-dependent nanodomain ordering, and provides important clues to the motives behind the enrichment of PI(4,5)P2 with polyunsaturated acyl-chains. We also show how Ca2+-induced PI(4,5)P2 nanodomains are able to generate local negative curvature, a phenomenon likely to play a role in membrane remodeling events. Phosphatidylinositol-4,5-bisphosphates are involved in membrane regulation and signalling in eukaryotes, with the distribution of acyl chains changing in response to stimuli. Here, molecular dynamics simulations and fluorescence spectroscopy experiments show that increasing acyl chain saturation increases the ordering of lipid nanodomains in artificial membranes.

Phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2) is a minor but ubiquitous component of the inner leaflet of the plasma membrane of eukaryotic cells. However, due to its particular complex biophysical properties, it stands out from its neighboring lipids as one of the most important regulators of membrane-associated signaling events. Despite its very low steady-state concentration, PI(4,5)P2 is able to engage in a multitude of simultaneous cellular functions that are temporally and spatially regulated through the presence of localized transient pools of PI(4,5)P2 in the membrane. These pools are crucial for the recruitment, activation, and organization of signaling proteins and consequent regulation of downstream signaling. The present review showcases some of the most important PI(4,5)P2 molecular and biophysical properties as well as their impact on its membrane dynamics, lateral organization, and interactions with other biochemical partners.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.