Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain.

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins1-3. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour3,4. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors5-9. Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P2, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.

In situ architecture of the ER-mitochondria encounter structure.

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.

The conformational plasticity of structurally unrelated lipid transport proteins correlates with their mode of action.

Lipid transfer proteins (LTPs) are key players in cellular homeostasis and regulation, as they coordinate the exchange of lipids between different cellular organelles. Despite their importance, our mechanistic understanding of how LTPs function at the molecular level is still in its infancy, mostly due to the large number of existing LTPs and to the low degree of conservation at the sequence and structural level. In this work, we use molecular simulations to characterize a representative dataset of lipid transport domains (LTDs) of 12 LTPs that belong to 8 distinct families. We find that despite no sequence homology nor structural conservation, the conformational landscape of LTDs displays common features, characterized by the presence of at least 2 main conformations whose populations are modulated by the presence of the bound lipid. These conformational properties correlate with their mechanistic mode of action, allowing for the interpretation and design of experimental strategies to further dissect their mechanism. Our findings indicate the existence of a conserved, fold-independent mechanism of lipid transfer across LTPs of various families and offer a general framework for understanding their functional mechanism.

Lipid scrambling is a general feature of protein insertases.

Glycerophospholipids are synthesized primarily in the cytosolic leaflet of the endoplasmic reticulum (ER) membrane and must be equilibrated between bilayer leaflets to allow the ER and membranes derived from it to grow. Lipid equilibration is facilitated by integral membrane proteins called "scramblases." These proteins feature a hydrophilic groove allowing the polar heads of lipids to traverse the hydrophobic membrane interior, similar to a credit card moving through a reader. Nevertheless, despite their fundamental role in membrane expansion and dynamics, the identity of most scramblases has remained elusive. Here, combining biochemical reconstitution and molecular dynamics simulations, we show that lipid scrambling is a general feature of protein insertases, integral membrane proteins which insert polypeptide chains into membranes of the ER and organelles disconnected from vesicle trafficking. Our data indicate that lipid scrambling occurs in the same hydrophilic channel through which protein insertion takes place and that scrambling is abolished in the presence of nascent polypeptide chains. We propose that protein insertases could have a so-far-overlooked role in membrane dynamics as scramblases.

Seipin accumulates and traps diacylglycerols and triglycerides in its ring-like structure.

Lipid droplets (LDs) are intracellular organelles responsible for lipid storage, and they emerge from the endoplasmic reticulum (ER) upon the accumulation of neutral lipids, mostly triglycerides (TG), between the two leaflets of the ER membrane. LD biogenesis takes place at ER sites that are marked by the protein seipin, which subsequently recruits additional proteins to catalyze LD formation. Deletion of seipin, however, does not abolish LD biogenesis, and its precise role in controlling LD assembly remains unclear. Here, we use molecular dynamics simulations to investigate the molecular mechanism through which seipin promotes LD formation. We find that seipin clusters TG, as well as its precursor diacylglycerol, inside its unconventional ring-like oligomeric structure and that both its luminal and transmembrane regions contribute to this process. This mechanism is abolished upon mutations of polar residues involved in protein-TG interactions into hydrophobic residues. Our results suggest that seipin remodels the membrane of specific ER sites to prime them for LD biogenesis.

Development of a coarse-grained model for surface-functionalized gold nanoparticles: towards an accurate description of their aggregation behavior.

Understanding the dispersion stability and aggregation propensity of self-assembled monolayer gold NPs at a molecular level is crucial to guide their rational design and to inform about the optimal surface functionalization for specific applications. To reach this goal, in silico modeling via coarse-grained (CG) molecular dynamics (MD) simulations is a fundamental tool to complement the information acquired from experimental studies since CG modeling allows to get a deep knowledge of the molecular interactions that take place at the nanoscale in this kind of systems. Unfortunately, current CG models of monolayer-protected AuNPs present several drawbacks that limit their accuracy in certain scenarios. We here develop a CG model that is fully compatible and extends the SPICA/SDK (Shinoda-DeVane-Klein) force field. Our model allows reproducing the behavior of AuNPs functionalized with hydrophobic as well as charged and more hydrophilic ligands. This model improves upon results obtained with previously derived CG force fields and successfully describes NPs aggregation and self-assembly in aqueous solution.

An in silico osmotic pressure approach allows characterization of pressure-area isotherms of lipid monolayers at low molecular areas.

Surface pressure-area isotherms of lipid monolayers at the air-water interface provide essential information about the structure and mechanical behaviour of lipid membranes. These curves can be readily obtained through Langmuir trough measurements and, as such, have been collected for decades in the field of membrane biochemistry. However, it is still challenging to directly observe and understand nanoscopic features of monolayers through such experiments, and molecular dynamics (MD) simulations are generally used to provide a molecular view of such interfaces. In MD simulations, the surface pressure-area (Π-A) isotherms are generally computed using the Kirkwood-Irving formula, that relies on the evaluation of the pressure tensor. This approach, however, has intrinsic limitations when the molecular area in the monolayer is low (typically < 60 Å2 per lipid). Recently, an alternative method to compute Π-A isotherms of surfactants, based on the calculation of the three-dimensional osmotic pressure via the implementation of semipermeable barriers was proposed. In this work, we investigate the feasibility of this approach for long-chain surfactants such as phospholipids. We identify some discrepancies between the computed values and experimental results, and we propose a semi-empirical correction based on the molecular structure of the surfactants at the monolayer interface. To validate the potential of this new approach, we simulate several phosphatidylcholine and phosphatidylethanolamine lipids at various temperatures using all-atom and coarse-grained force fields, and we compute the corresponding Π-A isotherms. Our results show that the Π-A isotherms obtained using the new method are in very good agreement with experiments and far superior to the canonical pressure tensor-based method at low molecular areas. This corrected osmotic pressure method allows for accurate characterization of the molecular packing in monolayers in various physical phases.

Estimating the accuracy of the MARTINI model towards the investigation of peripheral protein-membrane interactions.

Peripheral membrane proteins play a major role in numerous biological processes by transiently associating with cellular membranes, often with extreme membrane specificity. Because of the short-lived nature of these interactions, molecular dynamics (MD) simulations have emerged as an appealing tool to characterize at the structural level the molecular details of the protein-membrane interface. Transferable coarse-grained (CG) MD simulations, in particular, offer the possibility to investigate the spontaneous association of peripheral proteins with lipid bilayers of different compositions at limited computational cost, but they are hampered by the lack of a reliable a priori estimation of their accuracy and thus typically require a posteriori experimental validation. In this article, we investigate the ability of the MARTINI CG force field, specifically the 3 open-beta version, to reproduce known experimental observations regarding the membrane binding behavior of 12 peripheral membrane proteins and peptides. Based on observations of multiple binding and unbinding events in several independent replicas, we found that, despite the presence of false positives and false negatives, this model is mostly able to correctly characterize the membrane binding behavior of peripheral proteins, and to identify key residues found to disrupt membrane binding in mutagenesis experiments. While preliminary, our investigations suggest that transferable chemical-specific CG force fields have enormous potential in the characterization of the membrane binding process by peripheral proteins, and that the identification of negative results could help drive future force field development efforts.

Investigating the structural properties of hydrophobic solvent-rich lipid bilayers.

In vitro reconstitutions of lipid membranes have proven to be an indispensable tool to rationalize their molecular complexity and to understand their role in countless cellular processes. However, amongst the various techniques used to reconstitute lipid bilayers in vitro, several approaches are not solvent-free, but rather contain residual hydrophobic solvents in between the two bilayer leaflets, generally as a consequence of the procedure used to generate the bilayer. To what extent the presence of these hydrophobic solvents modifies bilayer properties with respect to native, solvent-free, conditions remains an open question that has important implications for the appropriate interpretation of numerous experimental observations. Here, we thorouhgly characterize hydrophobic solvent-rich lipid bilayers using atomistic molecular dynamics simulations. Our data indicate that while the presence of hydrophobic solvents at high concentrations, such as hexadecane, has a significant effect on membrane thickness, their effects on surface properties, membrane order and lateral stress are quite moderate. Our results corroborate the validity of in vitro approaches as model systems for the investigations of biological membranes but raise a few cautionary aspects that must be considered when investigating specific membrane properties.

Protonation Equilibrium in the Active Site of the Photoactive Yellow Protein.

The role and existence of low-barrier hydrogen bonds (LBHBs) in enzymatic and protein activity has been largely debated. An interesting case is that of the photoactive yellow protein (PYP). In this protein, two short HBs adjacent to the chromophore, p-coumaric acid (pCA), have been identified by X-ray and neutron diffraction experiments. However, there is a lack of agreement on the chemical nature of these H-bond interactions. Additionally, no consensus has been reached on the presence of LBHBs in the active site of the protein, despite various experimental and theoretical studies having been carried out to investigate this issue. In this work, we perform a computational study that combines classical and density functional theory (DFT)-based quantum mechanical/molecular mechanical (QM/MM) simulations to shed light onto this controversy. Furthermore, we aim to deepen our understanding of the chemical nature and dynamics of the protons involved in the two short hydrogen bonds that, in the dark state of PYP, connect pCA with the two binding pocket residues (E46 and Y42). Our results support the existence of a strong LBHB between pCA and E46, with the H fully delocalized and shared between both the carboxylic oxygen of E46 and the phenolic oxygen of pCA. Additionally, our findings suggest that the pCA interaction with Y42 can be suitably described as a typical short ionic H-bond of moderate strength that is fully localized on the phenolic oxygen of Y42.

Computational Approaches to Investigate and Design Lipid-binding Domains for Membrane Biosensing.

Association of proteins with cellular membranes is critical for signaling and membrane trafficking processes. Many peripheral lipid-binding domains have been identified in the last few decades and have been investigated for their specific lipid-sensing properties using traditional in vivo and in vitro studies. However, several knowledge gaps remain owing to intrinsic limitations of these methodologies. Thus, novel approaches are necessary to further our understanding in lipid-protein biology. This review briefly discusses lipid-binding domains that act as specific lipid biosensors and provides a broad perspective on the computational approaches such as molecular dynamics (MD) simulations and machine learning (ML)-based techniques that can be used to study protein-membrane interactions. We also highlight the need for de novo design of proteins that elicit specific lipid-binding properties.

Structure and Dynamics of the Acyl Chains in the Membrane Trafficking and Enzymatic Processing of Lipids.

The regulatory chemical mechanisms of lipid trafficking and degradation are involved in many pathophysiological processes, being implicated in severe pain, inflammation, and cancer. In addition, the processing of lipids is also relevant for industrial and environmental applications. However, there is poor understanding of the chemical features that control lipid membrane trafficking and allow lipid-degrading enzymes to efficiently select and hydrolyze specific fatty acids from a complex cellular milieu of bioactive lipids. This is particularly true for lipid acyl chains, which have diverse structures that can critically affect the many complex reactions needed to elongate, desaturate, or transport fatty acids. Building upon our own contributions in this field, we will discuss how molecular simulations, integrated with experimental evidence, have revealed that the structure and dynamics of the lipid tail are actively involved in modulating membrane trafficking at cellular organelles, and enzymatic reactions at cell membranes. Further evidence comes from recent crystal structures of lipid receptors and remodeling enzymes. Taken together, these recent works have identified those structural features of the lipid acyl chain that are crucial for the regioselectivity and stereospecificity of essential desaturation reactions. In this context, we will first illustrate how atomistic and coarse-grained simulations have elucidated the structure-function relationships between the chemical composition of the lipid's acyl chains and the molecular properties of lipid bilayers. Particular emphasis will be given to the prominent chemical role of the number of double carbon-carbon bonds along the lipid acyl chain, that is, discriminating between saturated, monounsaturated, and polyunsaturated lipids. Different levels of saturation in fatty acid molecules dramatically influence the biophysical properties of lipid assemblies and their interaction with proteins. We will then discuss the processing of lipids by membrane-bound enzymes. Our focus will be on lipids such as anandamide and 2-arachidonoylglycerol. These are the main molecules that act as neurotransmitters in the endocannabinoid system. Specifically, recent findings indicate a crucial interplay between the level of saturation of the lipid tail, its energetically and sterically favored conformations, and the hydrophobic accessory cavities in lipid-degrading enzymes, which help form catalytically active conformations of the selected substrate. This Account will emphasize how the specific chemical structure of acyl chains affects the molecular mechanisms for modulating membrane trafficking and selective hydrolysis. The results examined here show that, by using molecular simulations to investigate lipid plasticity and substrate flexibility, researchers can enrich their interpretation of experimental results about the structure-function relationships of lipids. This could positively impact chemical and biological studies in the field and ultimately support protein engineering studies and structure-based drug discovery to target lipid-processing enzymes.

An Atomistic Look into Bio-inspired Nanoparticles and their Molecular Interactions with Cells.

Nanoparticles (NPs) have sizes that approach those of pathogens and they can interact with the membranes of eukaryotic cells in an analogous fashion. Typically, NPs are taken up by the cell via the plasma membrane by receptor-mediated processes and subsequently interact with various endomembranes. Unlike pathogens, however, NPs lack the remarkable specificity gained during the evolutionary process and their design and optimization remains an expensive and time-consuming undertaking, especially considering the limited information available on their molecular interactions with cells. In this context, molecular dynamics (MD) simulations have emered as a promising strategy to investigate the mechanistic details of the interaction of NPs with mammalian or viral membranes. In particular, MD simulations have been extensively used to study the uptake process of NPs into the cell, focusing on membrane vesiculation, endocytic routes, or passive permeation processes. While such work is certainly relevant for understanding NP-cell interactions, it remains very difficult to determine the correspondence between generic models and the actual NP. Here, we review how chemically-specific MD simulations can provide rational guidelines towards further bio-inspired NP optimization.

PackMem: A Versatile Tool to Compute and Visualize Interfacial Packing Defects in Lipid Bilayers.

The analysis of the structural organization of lipid bilayers is generally performed across the direction normal to the bilayer/water interface, whereas the surface properties of the bilayer at the interface with water are often neglected. Here, we present PackMem, a bioinformatic tool that performs a topographic analysis of the bilayer surface from various molecular dynamics simulations. PackMem unifies and rationalizes previous analyses based on a Cartesian grid. The grid allows identification of surface regions defined as lipid-packing defects where lipids are loosely packed, leading to cavities in which aliphatic carbons are exposed to the solvent, either deep inside or close to the membrane surface. Examples are provided to show that the abundance of lipid-packing defects varies according to the temperature and to the bilayer composition. Because lipid-packing defects control the adsorption of peripheral proteins with hydrophobic insertions, PackMem is instrumental for us to understand and quantify the adhesive properties of biological membranes as well as their response to mechanical perturbations such as membrane deformation.

Understanding Conformational Dynamics of Complex Lipid Mixtures Relevant to Biology.

This is a perspective article entitled "Frontiers in computational biophysics: understanding conformational dynamics of complex lipid mixtures relevant to biology" which is following a CECAM meeting with the same name.

Interdigitation between Triglycerides and Lipids Modulates Surface Properties of Lipid Droplets.

Intracellular lipid droplets (LDs) are the main cellular site of metabolic energy storage. Their structure is unique inside the cell, with a core of esterified fatty acids and sterols, mainly triglycerides and sterol esters, surrounded by a single monolayer of phospholipids. Numerous peripheral proteins, including several that were previously associated with intracellular compartments surrounded by a lipid bilayer, have been recently shown to target the surface of LDs, but how they are able to selectively target this organelle remains largely unknown. Here, we use atomistic and coarse-grained molecular dynamics simulations to investigate the molecular properties of the LD surface and to characterize how it differs from that of a lipid bilayer. Our data suggest that although several surface properties are remarkably similar between the two structures, key differences originate from the interdigitation between surface phospholipids and core neutral lipids that occurs in LDs. This property is extremely sensitive to membrane undulations, unlike in lipid bilayers, and it strongly affects both lipid-packing defects and the lateral pressure profile. We observed a marked change in overall surface properties for surface tensions >10 mN/m, indicative of a bimodal behavior. Our simulations provide a comprehensive molecular characterization of the unique surface properties of LDs and suggest how the molecular properties of the surface lipid monolayer can be modulated by the underlying neutral lipids.

Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory.

Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.

Methyl-branched lipids promote the membrane adsorption of α-synuclein by enhancing shallow lipid-packing defects.

Alpha-synuclein (AS) is a synaptic protein that is directly involved in Parkinson's disease due to its tendency to form protein aggregates. Since AS aggregation can be dependent on the interactions between the protein and the cell plasma membrane, elucidating the membrane binding properties of AS is of crucial importance to establish the molecular basis of AS aggregation into toxic fibrils. Using a combination of in vitro reconstitution experiments based on Giant Unilamellar Vesicles (GUVs), confocal microscopy and all-atom molecular dynamics simulations, we have investigated the membrane binding properties of AS, with a focus on the relative contribution of hydrophobic versus electrostatic interactions. In contrast with previous observations, we did not observe any binding of AS to membranes containing the ganglioside GM1, even at relatively high GM1 content. AS, on the other hand, showed a stronger affinity for neutral flat membranes consisting of methyl-branched lipids. To rationalize these results, we used all-atom molecular dynamics simulations to investigate the influence of methyl-branched lipids on interfacial membrane properties. We found that methyl-branched lipids promote the membrane adsorption of AS by creating shallow lipid-packing defects to a larger extent than polyunsaturated and monounsaturated lipids. Our findings suggest that methyl-branched lipids may constitute a remarkably adhesive substrate for peripheral proteins that adsorb on membranes via hydrophobic insertions.

Origin of the spectral shifts among the early intermediates of the rhodopsin photocycle.

A combined strategy based on the computation of absorption energies, using the ZINDO/S semiempirical method, for a statistically relevant number of thermally sampled configurations extracted from QM/MM trajectories is used to establish a one-to-one correspondence between the structures of the different early intermediates (dark, batho, BSI, lumi) involved in the initial steps of the rhodopsin photoactivation mechanism and their optical spectra. A systematic analysis of the results based on a correlation-based feature selection algorithm shows that the origin of the color shifts among these intermediates can be mainly ascribed to alterations in intrinsic properties of the chromophore structure, which are tuned by several residues located in the protein binding pocket. In addition to the expected electrostatic and dipolar effects caused by the charged residues (Glu113, Glu181) and to strong hydrogen bonding with Glu113, other interactions such as π-stacking with Ala117 and Thr118 backbone atoms, van der Waals contacts with Gly114 and Ala292, and CH/π weak interactions with Tyr268, Ala117, Thr118, and Ser186 side chains are found to make non-negligible contributions to the modulation of the color tuning among the different rhodopsin photointermediates.