Molecular dynamics simulations of intracellular lipid droplets: a new tool in the toolbox.

Lipid droplets (LDs) are ubiquitous intracellular organelles with a central role in multiple lipid metabolic pathways. However, identifying correlations between their structural properties and their biological activity has proved challenging, owing to their unique physicochemical properties as compared with other cellular membranes. In recent years, molecular dynamics (MD) simulations, a computational methodology allowing the accurate description of molecular assemblies down to their individual components, have been demonstrated to be a useful and powerful approach for studying LD structural and dynamical properties. In this short review, we attempt to highlight, as comprehensively as possible, how MD simulations have contributed to our current understanding of multiple molecular mechanisms involved in LD biology.

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.

Improved Protein Model in SPICA Force Field.

The previous version of the SPICA coarse-grained (CG) force field (FF) protein model focused primarily on membrane proteins and successfully reproduced the dimerization free energies of several transmembrane helices and the stable structures of various membrane protein assemblies. However, that model had limited accuracy when applied to other proteins, such as intrinsically disordered proteins (IDPs) and peripheral proteins, because the dimensions of the IDPs in an aqueous solution were too compact, and protein binding on the lipid membrane surface was overstabilized. To improve the accuracy of the SPICA FF model for the simulation of such systems, in this study, we introduce protein secondary structure-dependent nonbonded interaction parameters to the backbone segments and reoptimize almost all nonbonded parameters for amino acids. The improved FF proposed here successfully reproduces the radii of gyration of various IDPs, the binding sensitivity of several peripheral membrane proteins, and the dimerization free energies of several transmembrane helices. The new model also shows improved agreement with experiments on the free energy of peptide association in water. In addition, an extensive library of nonbonded interactions between proteins and lipids, including various glycerophospholipids, sphingolipids, and cholesterol, allows the study of specific interactions between lipids and peripheral and transmembrane proteins. Hence, the new SPICA FF (version 2) proposed herein is applicable with high accuracy for simulating a wide range of protein systems.

Pragmatic Coarse-Graining of Proteins: Models and Applications.

The molecular details involved in the folding, dynamics, organization, and interaction of proteins with other molecules are often difficult to assess by experimental techniques. Consequently, computational models play an ever-increasing role in the field. However, biological processes involving large-scale protein assemblies or long time scale dynamics are still computationally expensive to study in atomistic detail. For these applications, employing coarse-grained (CG) modeling approaches has become a key strategy. In this Review, we provide an overview of what we call pragmatic CG protein models, which are strategies combining, at least in part, a physics-based implementation and a top-down experimental approach to their parametrization. In particular, we focus on CG models in which most protein residues are represented by at least two beads, allowing these models to retain some degree of chemical specificity. A description of the main modern pragmatic protein CG models is provided, including a review of the most recent applications and an outlook on future perspectives in the field.

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.

Malonyl-CoA is a conserved endogenous ATP-competitive mTORC1 inhibitor.

Cell growth is regulated by the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here we show that mTORC1 senses the capacity of a cell to synthesise fatty acids by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When fatty acid synthase (FASN) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with acetyl-CoA carboxylase 1 (ACC1) and FASN. Our findings represent a conserved and unique homeostatic mechanism whereby impaired fatty acid biogenesis leads to reduced mTORC1 activity to coordinately link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the existence of a physiological metabolite that directly inhibits the activity of a signalling kinase in mammalian cells by competing with ATP for binding.

Computational modeling of membrane trafficking processes: From large molecular assemblies to chemical specificity.

In the last decade, molecular dynamics (MD) simulations have become an essential tool to investigate the molecular properties of membrane trafficking processes, often in conjunction with experimental approaches. The combination of MD simulations with recent developments in structural biology, such as cryo-electron microscopy and artificial intelligence-based structure determination, opens new, exciting possibilities for future investigations. However, the full potential of MD simulations to provide a molecular view of the complex and dynamic processes involving membrane trafficking can only be realized if certain limitations are addressed, and especially those concerning the quality of coarse-grain models, which, despite recent successes in describing large-scale systems, still suffer from far-from-ideal chemical accuracy. In this review, we will highlight recent success stories of MD simulations in the investigation of membrane trafficking processes, their implications for future research, and the challenges that lie ahead in this specific research domain.

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.

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.

A conserved function of Human DLC3 and Drosophila Cv-c in testis development.

The identification of genes affecting gonad development is essential to understand the mechanisms causing Variations/Differences in Sex Development (DSD). Recently, a DLC3 mutation was associated with male gonadal dysgenesis in 46,XY DSD patients. We have studied the requirement of Cv-c, the Drosophila ortholog of DLC3, in Drosophila gonad development, as well as the functional capacity of DLC3 human variants to rescue cv-c gonad defects. We show that Cv-c is required to maintain testis integrity during fly development. We find that Cv-c and human DLC3 can perform the same function in fly embryos, as flies carrying wild type but not patient DLC3 variations can rescue gonadal dysgenesis, suggesting functional conservation. We also demonstrate that the StART domain mediates Cv-c's function in the male gonad independently from the GAP domain's activity. This work demonstrates a role for DLC3/Cv-c in male gonadogenesis and highlights a novel StART domain mediated function required to organize the gonadal mesoderm and maintain its interaction with the germ cells during testis development.

Monovalent ion-mediated charge-charge interactions drive aggregation of surface-functionalized gold nanoparticles.

Monolayer-protected metal nanoparticles (NPs) are not only promising materials with a wide range of potential industrial and biological applications, but they are also a powerful tool to investigate the behaviour of matter at nanoscopic scales, including the stability of dispersions and colloidal systems. This stability is dependent on a delicate balance between attractive and repulsive interactions that occur in the solution, and it is described in quantitative terms by the classic Derjaguin-Landau-Vewey-Overbeek (DLVO) theory, that posits that aggregation between NPs is driven by van der Waals interactions and opposed by electrostatic interactions. To investigate the limits of this theory at the nanoscale, where the continuum assumptions required by the DLVO theory break down, here we investigate NP dimerization by computing the Potential of Mean Force (PMF) of this process using fully atomistic MD simulations. Serendipitously, we find that electrostatic interactions can lead to the formation of metastable NP dimers at physiological ion concentrations. These dimers are stabilized by complexes formed by negatively charged ligands belonging to distinct NPs that are bridged by positively charged monovalent ions present in solution. We validate our findings by collecting tomographic EM images of NPs in solution and by quantifying their radial distribution function, that shows a marked peak at interparticle distance comparable with that of MD simulations. Taken together, our results suggest that not only van der Waals interactions, but also electrostatic interactions mediated by monovalent ions at physiological concentrations, contribute to attraction between nano-sized charged objects at very short length scales.

DNA Self-Assembly of Single Molecules with Deterministic Position and Orientation.

An ideal nanofabrication method should allow the organization of nanoparticles and molecules with nanometric positional precision, stoichiometric control, and well-defined orientation. The DNA origami technique has evolved into a highly versatile bottom-up nanofabrication methodology that fulfils almost all of these features. It enables the nanometric positioning of molecules and nanoparticles with stoichiometric control, and even the orientation of asymmetrical nanoparticles along predefined directions. However, orienting individual molecules has been a standing challenge. Here, we show how single molecules, namely, Cy5 and Cy3 fluorophores, can be incorporated in a DNA origami with controlled orientation by doubly linking them to oligonucleotide strands that are hybridized while leaving unpaired bases in the scaffold. Increasing the number of bases unpaired induces a stretching of the fluorophore linkers, reducing its mobility freedom, and leaves more space for the fluorophore to accommodate and find different sites for interaction with the DNA. Particularly, we explore the effects of leaving 0, 2, 4, 6, and 8 bases unpaired and find extreme orientations for 0 and 8 unpaired bases, corresponding to the molecules being perpendicular and parallel to the DNA double-helix, respectively. We foresee that these results will expand the application field of DNA origami toward the fabrication of nanodevices involving a wide range of orientation-dependent molecular interactions, such as energy transfer, intermolecular electron transport, catalysis, exciton delocalization, or the electromagnetic coupling of a molecule to specific resonant nanoantenna modes.

Triglyceride lipolysis triggers liquid crystalline phases in lipid droplets and alters the LD proteome.

Lipid droplets (LDs) are reservoirs for triglycerides (TGs) and sterol-esters (SEs), but how these lipids are organized within LDs and influence their proteome remain unclear. Using in situ cryo-electron tomography, we show that glucose restriction triggers lipid phase transitions within LDs generating liquid crystalline lattices inside them. Mechanistically this requires TG lipolysis, which decreases the LD's TG:SE ratio, promoting SE transition to a liquid crystalline phase. Molecular dynamics simulations reveal TG depletion promotes spontaneous TG and SE demixing in LDs, additionally altering the lipid packing of the PL monolayer surface. Fluorescence imaging and proteomics further reveal that liquid crystalline phases are associated with selective remodeling of the LD proteome. Some canonical LD proteins, including Erg6, relocalize to the ER network, whereas others remain LD-associated. Model peptide LiveDrop also redistributes from LDs to the ER, suggesting liquid crystalline phases influence ER-LD interorganelle transport. Our data suggests glucose restriction drives TG mobilization, which alters the phase properties of LD lipids and selectively remodels the LD proteome.

Recharging your fats: CHARMM36 parameters for neutral lipids triacylglycerol and diacylglycerol.

Neutral lipids (NLs) are an abundant class of cellular lipids. They are characterized by the total lack of charged chemical groups in their structure, and, as a consequence, they play a major role in intracellular lipid storage. NLs that carry a glycerol backbone, such as triacylglycerols (TGs) and diacylglycerols (DGs), are also involved in the biosynthetic pathway of cellular phospholipids, and they have recently been the subject of numerous structural investigations by means of atomistic molecular dynamics simulations. However, conflicting results on the physicochemical behavior of NLs were observed depending on the nature of the atomistic force field used. Here, we show that current phospholipid-derived CHARMM36 parameters for DGs and TGs cannot adequately reproduce interfacial properties of these NLs because of excessive hydrophilicity at the glycerol-ester region. By following a CHARMM36-consistent parameterization strategy, we develop improved parameters for both TGs and DGs that are compatible with both cutoff-based and particle mesh Ewald schemes for the treatment of Lennard-Jones interactions. We show that our improved parameters can reproduce interfacial properties of NLs and their behavior in more complex lipid assemblies. We discuss the implications of our findings in the context of intracellular lipid storage and NLs' cellular activity.

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.

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.