Molecular mechanisms and energetics of lipid droplet formation and directional budding.

The formation and budding of lipid droplets (LDs) are known to be governed by the LD size and by membrane tensions in the endoplasmic reticulum (ER) bilayer and LD-monolayers. Using coarse-grained simulations of an LD model, we first show that ER-embedded LDs of different sizes can form through a continuous transition from wide LD lenses to spherical LDs at a fixed LD size. The ER tendency to relax its bilayer modulates the transition via a subtle interplay between the ER and LD lipid densities. By calculating the energetic landscape of the LD transition, we demonstrate that this size-independent transition is regulated by the mechanical force balance of ER and LD-tensions, independent from membrane bending and line tension whose energetic contributions are negligible according to our calculations. Our findings explain experimental observation of stable LDs of various shapes. We then propose a novel mechanism for directional LD budding where the required membrane asymmetry is provided by the exchange of lipids between the LD-monolayers. Remarkably, we demonstrate that this budding process is energetically neutral. Consequently, LD budding can proceed by a modest energy input from proteins or other driving agents. We obtain equal lipid densities and membrane tensions in LD-monolayers throughout budding. Our findings indicate that unlike LD formation, LD budding by inter-monolayer lipid exchange is a tension-independent process.

Membrane-Mediated Cooperative Interactions of CD47 and SIRPα.

The specific binding of the ubiquitous 'marker of self' protein CD47 to the SIRPα protein anchored in the macrophage plasma membrane results in the inhibition of the engulfment of 'self' cells by macrophages and thus constitutes a key checkpoint of our innate immune system. Consequently, the CD47-SIRPα protein complex has been recognized as a potential therapeutic target in cancer and inflammation. Here, we introduce a lattice-based mesoscale model for the biomimetic system studied recently in fluorescence microscopy experiments where GFP-tagged CD47 proteins on giant plasma membrane vesicles bind to SIRPα proteins immobilized on a surface. Computer simulations of the lattice-based mesoscale model allow us to study the biomimetic system on multiple length scales, ranging from single nanometers to several micrometers and simultaneously keep track of single CD47-SIRPα binding and unbinding events. Our simulations not only reproduce data from the fluorescence microscopy experiments but also are consistent with results of several other experiments, which validates our numerical approach. In addition, our simulations yield quantitative predictions on the magnitude and range of effective, membrane-mediated attraction between CD47-SIRPα complexes. Such detailed information on CD47-SIRPα interactions cannot be obtained currently from experiments alone. Our simulation results thus extend the present understanding of cooperative effects in CD47-SIRPα interactions and may have an influence on the advancement of new cancer treatments.

Effects of biophysical membrane properties on recognition of phosphatidylserine, or phosphatidylinositol 4-phosphate by lipid biosensors LactC2, or P4M.

Lipid biosensors are molecular tools used both in vivo and in vitro applications, capable of selectively detecting specific types of lipids in biological membranes. However, despite their extensive use, there is a lack of systematic characterization of their binding properties in various membrane conditions. The purpose of this study was to investigate the impact of membrane properties, such as fluidity and membrane charge, on the sensitivity of two lipid biosensors, LactC2 and P4M, to their target lipids, phosphatidylserine (PS) or phosphatidylinositol 4-phosphate (PI4P), respectively. Dual-color fluorescence cross-correlation spectroscopy, employed in this study, provided a useful technique to investigate interactions of these recombinant fluorescent biosensors with liposomes of varying compositions. The results of the study demonstrate that the binding of the LactC2 biosensor to low levels of PS in the membrane is highly supported by the presence of anionic lipids or membrane fluidity. However, at high PS levels, the presence of anionic lipids does not further enhance binding of LactC2. In contrast, neither membrane charge, nor membrane fluidity significantly affect the binding affinity of P4M to PI4P. These findings provide valuable insights into the role of membrane properties on the binding properties of lipid biosensors.

Crystal Structure of the ORP8 Lipid Transport ORD Domain: Model of Lipid Transport.

ORPs are lipid-transport proteins belonging to the oxysterol-binding protein family. They facilitate the transfer of lipids between different intracellular membranes, such as the ER and plasma membrane. We have solved the crystal structure of the ORP8 lipid transport domain (ORD8). The ORD8 exhibited a ß-barrel fold composed of anti-parallel ß-strands, with three α-helices replacing ß-strands on one side. This mixed alpha-beta structure was consistent with previously solved structures of ORP2 and ORP3. A large cavity (≈1860 Å3) within the barrel was identified as the lipid-binding site. Although we were not able to obtain a lipid-bound structure, we used computer simulations based on our crystal structure to dock PS and PI4P molecules into the putative lipid-binding site of the ORD8. Comparative experiments between the short ORD8ΔLid (used for crystallography) and the full-length ORD8 (lid containing) revealed the lid's importance for stable lipid binding. Fluorescence assays revealed different transport efficiencies for PS and PI4P, with the lid slowing down transport and stabilizing cargo. Coarse-grained simulations highlighted surface-exposed regions and hydrophobic interactions facilitating lipid bilayer insertion. These findings enhance our comprehension of ORD8, its structure, and lipid transport mechanisms, as well as provide a structural basis for the design of potential inhibitors.

Leaflet Tensions Control the Spatio-Temporal Remodeling of Lipid Bilayers and Nanovesicles.

Biological and biomimetic membranes are based on lipid bilayers, which consist of two monolayers or leaflets. To avoid bilayer edges, which form when the hydrophobic core of such a bilayer is exposed to the surrounding aqueous solution, a single bilayer closes up into a unilamellar vesicle, thereby separating an interior from an exterior aqueous compartment. Synthetic nanovesicles with a size below 100 nanometers, traditionally called small unilamellar vesicles, have emerged as potent platforms for the delivery of drugs and vaccines. Cellular nanovesicles of a similar size are released from almost every type of living cell. The nanovesicle morphology has been studied by electron microscopy methods but these methods are limited to a single snapshot of each vesicle. Here, we review recent results of molecular dynamics simulations, by which one can monitor and elucidate the spatio-temporal remodeling of individual bilayers and nanovesicles. We emphasize the new concept of leaflet tensions, which control the bilayers' stability and instability, the transition rates of lipid flip-flops between the two leaflets, the shape transformations of nanovesicles, the engulfment and endocytosis of condensate droplets and rigid nanoparticles, as well as nanovesicle adhesion and fusion. To actually compute the leaflet tensions, one has to determine the bilayer's midsurface, which represents the average position of the interface between the two leaflets. Two particularly useful methods to determine this midsurface are based on the density profile of the hydrophobic lipid chains and on the molecular volumes.

Membrane curvature sensing by model biomolecular condensates.

Biomolecular condensates (BCs) are fluid droplets that form in biological cells by liquid-liquid phase separation. Their major components are intrinsically disordered proteins. Vast attention has been given in recent years to BCs inside the cytosol and nucleus. BCs at the cell membrane have not been studied to the same extent so far. However, recent studies provide increasingly more examples of interfaces between BCs and membranes which function as platforms for diverse biomolecular processes. Galectin-3, for example, is known to mediate clathrin-independent endocytosis and has been recently shown to undergo liquid-liquid phase separation, but the function of BCs of galectin-3 in endocytic pit formation is unknown. Here, we use dissipative particle dynamics simulations to study a generic coarse-grained model for BCs interacting with lipid membranes. In analogy to galectin-3, we consider polymers comprising two segments - one of them mediates multivalent attractive interactions between the polymers, and the other one has affinity for association with specific lipid head groups. When these polymers are brought into contact with a multi-component membrane, they spontaneously assemble into droplets and, simultaneously, induce lateral separation of lipids within the membrane. Interestingly, we find that if the membrane is bent, the polymer droplets localize at membrane regions curved inward. Although the polymers have no particular shape or intrinsic curvature, they appear to sense membrane curvature when clustered at the membrane. Our results indicate toward a generic mechanism of membrane curvature sensing by BCs involved in such processes as endocytosis.

Molecular Dynamics Simulations of Immune Receptors and Ligands.

Molecular dynamics simulations of immune receptor and ligand proteins in their native membrane environment allow to determine the orientational and structural variability of the proteins and protein complexes. The simulations complement the static, "membrane-free" structural information obtained from cryo-EM structures of transmembrane proteins in detergent micelles or from crystal structures of extracellular protein domains. Here we describe how to set up and perform simulations of transmembrane receptors, ligands, and receptor-ligand complexes.

Mechanical forces impair antigen discrimination by reducing differences in T-cell receptor/peptide-MHC off-rates.

T cells use their T-cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains debated. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity TCR/pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds). This was confirmed by molecular dynamics simulations. Consistent with these findings, we show that the best-characterized catch bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination, and we suggest a role for the adhesion receptors CD2 and LFA-1 in force-shielding the TCR/pMHC interaction.

Interplay of receptor-ligand binding and lipid domain formation during cell adhesion.

Cell adhesion involved in biological processes such as cell migration, immune responses, and cancer metastasis, is mediated by the specific binding of receptor and ligand proteins. Some of these proteins exhibit affinity for nanoscale lipid clusters in cell membranes. A key question is how these nanoscale lipid clusters influence and react to the receptor-ligand binding during cell adhesion. In this article, we review recent computational studies that shed new light on the interplay of the receptor-ligand binding and the formation of lipid domains in adhering membranes. These studies indicate that the receptor-ligand binding promotes coalescence of lipid clusters into mesoscale domains, which, in turn, enhances both the affinity and cooperativity of the receptor-ligand binding in cell-cell adhesion with mobile ligands. In contrast, in the case of cell-extracellular matrix adhesion with immobile ligands, the receptor-ligand binding and the lipid cluster coalescence can be correlated or anti-correlated, depending strongly on the ligand distribution. These findings deepen our understanding of correlations between cell adhesion and membrane heterogeneities.

Conformational ensemble of the full-length SARS-CoV-2 nucleocapsid (N) protein based on molecular simulations and SAXS data.

The nucleocapsid protein of the SARS-CoV-2 virus comprises two RNA-binding domains and three regions that are intrinsically disordered. While the structures of the RNA-binding domains have been solved using protein crystallography and NMR, current knowledge of the conformations of the full-length nucleocapsid protein is rather limited. To fill in this knowledge gap, we combined coarse-grained molecular simulations with data from small-angle X-ray scattering (SAXS) experiments using the ensemble refinement of SAXS (EROS) method. Our results show that the dimer of the full-length nucleocapsid protein exhibits large conformational fluctuations with its radius of gyration ranging from about 4 to 8 nm. The RNA-binding domains do not make direct contacts. The disordered region that links these two domains comprises a hydrophobic α-helix which makes frequent and nonspecific contacts with the RNA-binding domains. Each of the intrinsically disordered regions adopts conformations that are locally compact, yet on average, much more extended than Gaussian chains of equivalent lengths. We offer a detailed picture of the conformational ensemble of the nucleocapsid protein dimer under near-physiological conditions, which will be important for understanding the nucleocapsid assembly process.

Mapping the deformability of natural and designed cellulosomes in solution.

BACKGROUND: Natural cellulosome multi-enzyme complexes, their components, and engineered 'designer cellulosomes' (DCs) promise an efficient means of breaking down cellulosic substrates into valuable biofuel products. Their broad uptake in biotechnology relies on boosting proximity-based synergy among the resident enzymes, but the modular architecture challenges structure determination and rational design. RESULTS: We used small angle X-ray scattering combined with molecular modeling to study the solution structure of cellulosomal components. These include three dockerin-bearing cellulases with distinct substrate specificities, original scaffoldins from the human gut bacterium Ruminococcus champanellensis (ScaA, ScaH and ScaK) and a trivalent cohesin-bearing designer scaffoldin (Scaf20L), followed by cellulosomal complexes comprising these components, and the nonavalent fully loaded Clostridium thermocellum CipA in complex with Cel8A from the same bacterium. The size analysis of Rg and Dmax values deduced from the scattering curves and corresponding molecular models highlight their variable aspects, depending on composition, size and spatial organization of the objects in solution. CONCLUSIONS: Our data quantifies variability of form and compactness of cellulosomal components in solution and confirms that this native plasticity may well be related to speciation with respect to the substrate that is targeted. By showing that scaffoldins or components display enhanced compactness compared to the free objects, we provide new routes to rationally enhance their stability and performance in their environment of action.

Osh6 Revisited: Control of PS Transport by the Concerted Actions of PI4P and Sac1 Phosphatase.

Osh6, a member of the oxysterol-binding protein-related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.

Structural variability and concerted motions of the T cell receptor - CD3 complex.

We investigate the structural and orientational variability of the membrane-embedded T cell receptor (TCR) - CD3 complex in extensive atomistic molecular dynamics simulations based on the recent cryo-EM structure determined by Dong et al., 2019. We find that the TCR extracellular (EC) domain is highly variable in its orientation by attaining tilt angles relative to the membrane normal that range from 15° to 55°. The tilt angle of the TCR EC domain is both coupled to a rotation of the domain and to characteristic changes throughout the TCR - CD3 complex, in particular in the EC interactions of the Cß FG loop of the TCR, as well as in the orientation of transmembrane helices. The concerted motions of the membrane-embedded TCR - CD3 complex revealed in our simulations provide atomistic insights on conformational changes of the complex in response to tilt-inducing forces on antigen-bound TCRs.

Cooperative Stabilization of Close-Contact Zones Leads to Sensitivity and Selectivity in T-Cell Recognition.

T cells are sensitive to 1 to 10 foreign-peptide-MHC complexes among a vast majority of self-peptide-MHC complexes, and discriminate selectively between peptide-MHC complexes that differ not much in their binding affinity to T-cell receptors (TCRs). Quantitative models that aim to explain this sensitivity and selectivity largely focus on single TCR/peptide-MHC complexes, but T cell adhesion involves a multitude of different complexes. In this article, we demonstrate in a three-dimensional computational model of T-cell adhesion that the cooperative stabilization of close-contact zones is sensitive to one to three foreign-peptide-MHC complexes and occurs at a rather sharp threshold affinity of these complexes, which implies selectivity. In these close-contact zones with lateral extensions of hundred to several hundred nanometers, few TCR/foreign-peptide-MHC complexes and many TCR/self-peptide-MHC complexes are segregated from LFA-1/ICAM-1 complexes that form at larger membrane separations. Previous high-resolution microscopy experiments indicate that the sensitivity and selectivity in the formation of closed-contact zones reported here are relevant for T-cell recognition, because the stabilization of close-contact zones by foreign, agonist peptide-MHC complexes precedes T-cell signaling and activation in the experiments.

Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization.

Cellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule fluorescence resonance energy transfer and molecular simulations on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.

Interplay between cooperativity of intercellular receptor-ligand binding and coalescence of nanoscale lipid clusters in adhering membranes.

Adhesion of biological cells is mediated by the specific binding of receptors and ligands which are typically large proteins spanning through the plasma membranes of the contacting cells. The receptors and ligands can exhibit affinity for nanoscale lipid clusters that form within the plasma membrane. A central question is how these nanoscale lipid clusters physically affect and respond to the receptor-ligand binding during cell adhesion. Within the framework of classical statistical mechanics we find that the receptor-ligand binding reduces the threshold energy for lipid clusters to coalesce into mesoscale domains by up to ∼50%, and that the formation of these domains induces significant cooperativity of the receptor-ligand binding. The interplay between the receptor-ligand binding cooperativity and the lipid domain formation manifests acute sensitivity of the membrane system to changes in control parameters. This sensitivity can be crucial in cell signaling and immune responses.

Capturing the Conformational Ensemble of the Mixed Folded Polyglutamine Protein Ataxin-3.

Ataxin-3 is a deubiquitinase involved in protein quality control and other essential cellular functions. It preferentially interacts with polyubiquitin chains of four or more units attached to proteins delivered to the ubiquitin-proteasome system. Ataxin-3 is composed of an N-terminal Josephin domain and a flexible C terminus that contains two or three ubiquitin-interacting motifs (UIMs) and a polyglutamine tract, which, when expanded beyond a threshold, leads to protein aggregation and misfolding and causes spinocerebellar ataxia type 3. The high-resolution structure of the Josephin domain is available, but the structural and dynamical heterogeneity of ataxin-3 has so far hindered the structural description of the full-length protein. Here, we characterize non-expanded and expanded variants of ataxin-3 in terms of conformational ensembles adopted by the proteins in solution by jointly using experimental data from nuclear magnetic resonance and small-angle X-ray scattering with coarse-grained simulations. Our results pave the way to a molecular understanding of polyubiquitin recognition.

Pseudo-Improper-Dihedral Model for Intrinsically Disordered Proteins.

We present a new coarse-grained Cα-based protein model with a nonradial multibody pseudo-improper-dihedral potential that is transferable, time-independent, and suitable for molecular dynamics. It captures the nature of backbone and side-chain interactions between amino acid residues by adapting a simple improper dihedral term for a one-bead-per-residue model. It is parameterized for intrinsically disordered proteins and applicable to simulations of such proteins and their assemblies on millisecond time scales.

Intercellular Receptor-Ligand Binding and Thermal Fluctuations Facilitate Receptor Aggregation in Adhering Membranes.

Nanoscale molecular clusters in cell membranes can serve as platforms to recruit membrane proteins for various biological functions. A central question is how these nanoclusters respond to physical contacts between cells. Using a statistical mechanics model and Monte Carlo simulations, we explore how the adhesion of cell membranes affects the stability and coalescence of clusters enriched in receptor proteins. Our results show that intercellular receptor-ligand binding and membrane shape fluctuations can lead to receptor aggregation within the adhering membranes even if large-scale clusters are thermodynamically unstable in nonadhering membranes.

Structural insights into Acyl-coenzyme A binding domain containing 3 (ACBD3) protein hijacking by picornaviruses.

Many picornaviruses hijack the Golgi resident Acyl-coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4-kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4-phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small-angle X-ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.