Abstract
SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike ...extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.
The long-range communication between the two exosites of human α-thrombin (thrombin) tightly modulates the protein-effector interactions. Duplex/quadruplex aptamers represent an emerging class of ...very effective binders of thrombin. Among them, NU172 and HD22 aptamers are at the forefront of exosite I and II recognition, respectively. The present study investigates the simultaneous binding of these two aptamers by combining a structural and dynamics approach. The crystal structure of the ternary complex formed by the thrombin with NU172 and HD22_27mer provides a detailed view of the simultaneous binding of these aptamers to the protein, inspiring the design of novel bivalent thrombin inhibitors. The crystal structure represents the starting model for molecular dynamics studies, which point out the cooperation between the binding at the two exosites. In particular, the binding of an aptamer to its exosite reduces the intrinsic flexibility of the other exosite, that preferentially assumes conformations similar to those observed in the bound state, suggesting a predisposition to interact with the other aptamer. This behaviour is reflected in a significant increase of the anticoagulant activity of NU172 when the inactive HD22_27mer is bound to exosite II, providing a clear evidence of the synergic action of the two aptamers.
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•The simultaneous binding of the NU172/HD22_27mer aptamers to thrombin is unravelled.•MD studies provide clear insights on the cross-talk between thrombin exosites.•The in vitro synergic effect of NU172/HD22_27mer in anticoagulation is demonstrated.•Present findings provide clues on the design of polyvalent antithrombin aptamers.
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Inositol phospholipids are well known to form clusters in the cytoplasmic leaflet of the plasma membrane that are responsible for the interaction and recruitment of proteins involved ...in key biological processes like endocytosis, ion channel activation and secondary messenger production. Although their phosphorylated inositol ring headgroup plays an important role in protein binding, its orientation with respect to the plane of the membrane and its lateral packing density has not been previously described experimentally.
Here, we study phosphatidylinositol 4,5-bisphosphate (PIP2) planar model membranes in the form of Langmuir monolayers by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry to elucidate the relation between lateral (in-plane) and perpendicular (out-of-plane) molecular organization of PIP2.
Different surface areas were explored through monolayer compression, allowing us to correlate the formation of transient PIP2 clusters with the change in orientation of the inositol-biphosphate headgroup, which was experimentally determined by neutron reflectometry.
Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A ...thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin–echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.
Cartoons of the mixed monolayers of GO and DPPC and evolution of the volume fraction of each component with the surface pressure.
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Graphene oxide-based nanotechnology has aroused a ...great interest due to its applications in the biomedical and optoelectronic fields. The wide use of these materials makes it necessary to study its potential toxicity associated with the inhalation of Graphene Oxide (GO) nanoparticles and its interaction with the lung surfactant. Langmuir monolayers have proven to be an excellent tool for studying the properties of the lung surfactant and the effect of intercalation of nanoparticles on its structure and properties. Therefore, to know the origin of the phospholipids/GO interaction and the structure of the lipid layer with GO, in this work we study the effect of the insertion of GO sheets on a Langmuir film of 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC).
Surface pressure-area isotherms, Neutron (NR) and X-ray Reflectivity (XRR) and Grazing Incidence X-ray Diffraction (GIXD) measurements of hydrogenated and deuterated DPPC monolayers with and without GO have been carried out.
The results outline a strong interaction between the GO and the zwitterionic form of DPPC and prove that GO is in three regions of the DPPC monolayer, the aliphatic chains of DPPC, the head groups and water in the subphase. Comparison between results obtained with hydrogenated and deuterated DPPC allows concluding that both, electrostatic attractions, and dispersion forces are responsible of the interaction GO/DPPC. Results also demonstrated that the insertion of GO into the DPPC aliphatic chains does not induce significant changes on unit cell of DPPC.
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Unravelling the structural diversity of cellular membranes is a paramount challenge in life sciences. In particular, lipid composition affects the membrane collective behaviour, and ...its interactions with other biological molecules.
Here, the relationship between membrane composition and resultant structural features was investigated by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry on in vitro membrane models of the mammalian plasma and endoplasmic-reticulum-Golgi intermediate compartment membranes in the form of Langmuir monolayers. Natural extracted yeast lipids were used because, unlike synthetic lipids, the acyl chain saturation pattern of yeast and mammalian lipids are similar.
The structure of the model membranes, orthogonal to the plane of the membrane, as well as their lateral packing, were found to depend strongly on their specific composition, with cholesterol having a major influence on the in-plane morphology, yielding a coexistence of liquid-order and liquid-disorder phases.
The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force ...microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological membranes with the incorporation of inositol phospholipids and synthetic endocytic motifs.
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•Characterization of model lipid bilayers enriched in PIP2 and CD4 lipopeptide.•Neutron Reflectometry, AFM, and QCM-D reveal mechanical and structural properties.•Divalent cations enhance PIP2 liposome adsorption and fusion on solid substrate.•PIP2-CD4 interaction affects the membrane curvature.
Langmuir monolayers of 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, known as DPPC, at the air/water interface are extensively used as model systems of biomembranes and pulmonary surfactant. The ...properties of these monolayers have been mainly investigated by surface pressure–area isotherms coupled with different complementary techniques such as Brewster angle microscopy, for example. Several attempts using neutron reflectometry (NR) or ellipsometry have also appeared in the literature. Here, we report structural information obtained by using NR and ellipsometry on DPPC monolayers in the liquid condensed phase. On one side, NR can resolve the thickness of the aliphatic tails and the degree of hydration of the polar headgroups. On the other side, ellipsometry gives information on the refractive index and, therefore, on the physical state of the monolayer. The thickness and surface excess obtained by multiple-angle-of-incidence ellipsometry (MAIE) is compared with the results from NR measurements yielding a good agreement. Besides, a novel approach is reported to calculate the optical anisotropy of the DPPC monolayer that depends on the orientation of the aliphatic chains. The results from both NR and ellipsometry are also discussed in the context of the existing results for DPPC monolayers at the air/water interface. The differences observed are rationalized by the presence of buffer molecules interacting with phospholipids.
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