Two-Dimensional Interfacial Exchange Diffusion Has the Potential to Augment Spatiotemporal Precision of Ca2+ Signaling

Nano-junctions between the endoplasmic reticulum and cytoplasmic surfaces of the plasma membrane and other organelles shape the spatiotemporal features of biological Ca2+ signals. Herein, we propose that 2D Ca2+ exchange diffusion on the negatively charged phospholipid surface lining nano-junctions participates in guiding Ca2+ from its source (channel or carrier) to its target (transport protein or enzyme). Evidence provided by in vitro Ca2+ flux experiments using an artificial phospholipid membrane is presented in support of the above proposed concept, and results from stochastic simulations of Ca2+ trajectories within nano-junctions are discussed in order to substantiate its possible requirements. Finally, we analyze recent literature on Ca2+ lipid interactions, which suggests that 2D interfacial Ca2+ diffusion may represent an important mechanism of signal transduction in biological systems characterized by high phospholipid surface to aqueous volume ratios.

Post-Translational Modifications Optimize the Ability of SARS-CoV-2 Spike for Effective Interaction with Host Cell Receptors

SARS-CoV2 spike glycoprotein is prime target for vaccines and for diagnostics and therapeutic antibodies against the virus. While anchored in the viral envelope, for effective virulance, the spike needs to maintain structural flexibility to recognize the host cell surface receptors and bind to them, a property that can heavily hinge upon the dynamics of the unresolved domains, most prominently the stalk. Construction of the complete, membrane-bound spike model and the description of its dynamics remain critical steps in understanding the inner working of this key element in viral infection. Using a hybrid approach, combining homology modeling, protein-protein docking and MD simulations, guided by biochemical and glycomics data, we have developed a full-length, membrane-bound, palmitoylated and fully-glycosylated spike structure in a native membrane. Multi-microsecond MD simulations of this model, the longest known trajectory of the full-spike, reveals conformational dynamics employed by the protein to explore the crowded surface of the host cell. In agreement with cryoEM, three flexiblele hinges in stalk allow for global conformational heterogeneity of spike in the fully-glycosyslated system mediated by glycan-glycan and glycan-lipid interactions. Dynamical range of spike is considerably reduced in its non-glycosylated form, confining the area explored by the spike on the host cell surface. Furthermore, palmitoylation of the membrane domain amplify the local curvature that may prime the fusion. We show that the identified hinge regions are highly conserved in SARS coronaviruses, highlighting their functional importance in enhancing viral infection, and thereby provide novel points for discovery of alternative therapeutics against the virus.SignificanceSARS-CoV2 Spike protein, which forms the basis for high pathogenicity and transmissibility of the virus, is also prime target for the development of both diagnostics and vaccines for the debilitating disease caused by the virus. We present a full model of spike methodically crafted and used to study its atomic-level dynamics by multiple-µs simulations. The results shed new light on the impact of posttranslational modifications in the pathogenicity of the virus. We show how glycan-glycan and glycan-lipid interactions broaden the protein’s dynamical range, and thereby, its effective interaction with the surface receptors on the host cell. Palmitoylation of spike membrane domain, on the other hand, results in a unique deformation pattern that might prime the membrane for fusion.

Entropy in the molecular recognition of membrane protein-lipid interactions

Understanding the molecular driving forces that underlie membrane protein-lipid interactions requires the characterization of their binding thermodynamics. Here, we employ native mass spectrometry in conjunction with a variable temperature apparatus to determine the thermodynamics of individual lipid binding events to the human G-protein-gated inward rectifier potassium channel, Kir3.2. We find that Kir3.2 displays distinct thermodynamic strategies to engage phosphatidylinositol (PI) and phosphorylated forms thereof. The addition of a 4’- phosphate to PI with 18:1-18:1 (DO) tails results in an increase in favorable entropy along with an enthalpic penalty. The binding of PI with two or more phosphates is more complex where lipids bind to Kir3.2 with the cytoplasmic domain in either a docked or extended configuration. Remarkably, the interaction of 4,5-bisphosphate DOPI (DOPI(4,5)P2) with Kir3.2 is solely driven by a large, favorable change in entropy. Installment of a third 3’-phosphate to DOPI(4,5)P2 results in an alternative thermodynamic strategy for the first binding event whereas each successive binding event shows strong enthalpy-entropy compensation. PI(4,5)P2 with 18:0-20:4 tails results in an inversion of thermodynamic parameters where the change in enthalpy now dominates. Collectively, the data show that entropy can indeed play important roles in regulating membrane protein-lipid interactions.

Lipid Interactions Between Flaviviruses and Mosquito Vectors

Mosquito-borne flaviviruses, such as dengue (DENV), Zika (ZIKV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses, threaten a large part of the human populations. In absence of therapeutics and effective vaccines against each flaviviruses, targeting viral metabolic requirements in mosquitoes may hold the key to new intervention strategies. Development of metabolomics in the last decade opened a new field of research: mosquito metabolomics. It is now clear that flaviviruses rely on mosquito lipids, especially phospholipids, for their cellular cycle and propagation. Here, we review the biosyntheses of, biochemical properties of and flaviviral interactions with mosquito phospholipids. Phospholipids are structural lipids with a polar headgroup and apolar acyl chains, enabling the formation of lipid bilayer that form plasma- and endomembranes. Phospholipids are mostly synthesized through the de novo pathway and remodeling cycle. Variations in headgroup and acyl chains influence phospholipid physicochemical properties and consequently the membrane behavior. Flaviviruses interact with cellular membranes at every step of their cellular cycle. Recent evidence demonstrates that flaviviruses reconfigure the phospholipidome in mosquitoes by regulating phospholipid syntheses to increase virus multiplication. Identifying the phospholipids involved and understanding how flaviviruses regulate these in mosquitoes is required to design new interventions.

TMEM16 scramblases thin the membrane to enable lipid scrambling

TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. It was proposed that lipid headgroups move between leaflets through a membrane-spanning hydrophilic groove. Direct information on lipid-groove interactions is lacking. We report the 2.3 Å resolution cryoEM structure of the Ca2+-bound afTMEM16 scramblase in nanodiscs showing how rearrangement of individual lipids at the open pathway results in pronounced membrane thinning. Only the groove’s intracellular vestibule contacts lipids, and mutagenesis suggests scrambling does not entail specific protein-lipid interactions with the extracellular vestibule. Further, we find scrambling can occur outside a closed groove in thinner membranes and is inhibited in thicker membranes despite an open pathway. Our results show how afTMEM16 thins the membrane to enable scrambling and that an open hydrophilic pathway is not a structural requirement to allow rapid transbilayer movement of lipids. This mechanism could be extended to other scramblases lacking a hydrophilic groove.