Martini 3: a general purpose force field for coarse-grained molecular dynamics.

The coarse-grained Martini force field is widely used in biomolecular simulations. Here we present the refined model, Martini 3 ( http://cgmartini.nl ), with an improved interaction balance, new bead types and expanded ability to include specific interactions representing, for example, hydrogen bonding and electronic polarizability. The updated model allows more accurate predictions of molecular packing and interactions in general, which is exemplified with a vast and diverse set of applications, ranging from oil/water partitioning and miscibility data to complex molecular systems, involving protein-protein and protein-lipid interactions and material science applications as ionic liquids and aedamers.

Effect of palmitoylation on the dimer formation of the human dopamine transporter.

The human dopamine transporter (hDAT) is one in three members of the monoamine transporter family (MAT). hDAT is essential for regulating the dopamine concentration in the synaptic cleft through dopamine reuptake into the presynaptic neuron; thereby controlling hDAT dopamine signaling. Dysfunction of the transporter is linked to several psychiatric disorders. hDAT and the other MATs have been shown to form oligomers in the plasma membrane, but only limited data exists on which dimeric and higher order oligomeric states are accessible and energetically favorable. In this work, we present several probable dimer conformations using computational coarse-grained self-assembly simulations and assess the relative stability of the different dimer conformations using umbrella sampling replica exchange molecular dynamics. Overall, the dimer conformations primarily involve TM9 and/or TM11 and/or TM12 at the interface. Furthermore, we show that a palmitoyl group (palm) attached to hDAT on TM12 modifies the free energy of separation for interfaces involving TM12, suggesting that S-palmitoylation may change the relative abundance of dimers involving TM12 in a biological context. Finally, a comparison of the identified interfaces of hDAT and palmitoylated hDAT to the human serotonin transporter interfaces and the leucine transporter interface, suggests similar dimer conformations across these protein family.

Molecular Modeling Investigation of the Interaction between Humicola insolens Cutinase and SDS Surfactant Suggests a Mechanism for Enzyme Inactivation.

One of the largest commercial applications of enzymes and surfactants is as main components in modern detergents. The high concentration of surfactant compounds usually present in detergents can, however, negatively affect the enzymatic activity. To remedy this drawback, it is of great importance to characterize the interaction between the enzyme and the surfactant molecules at an atomistic resolution. The protein enzyme cutinase from the thermophilic and saprophytic fungus called Humicola insolens (HiC) is a promising candidate for use in detergents thanks to its hydrolase activity targeting mostly biopolyesters (e.g., cutin). HiC is, however, inhibited by low concentrations of sodium dodecyl sulfate (SDS), an ubiquitous surfactant. In this work, we investigate the interaction between HiC and SDS using molecular dynamics simulations. Simulations of HiC dissolved in different aqueous concentrations of SDS show the interaction between HiC and SDS monomers, as well as the formation and dynamics of SDS micelles on the surface of the enzyme. These results suggest a mechanism of cutinase inhibition by SDS, which involves the nucleation of aggregates of SDS molecules on hydrophobic patches on the cutinase surface. Notably, a primary binding site for monomeric SDS is identified near the active site of HiC constituting a possible nucleation point for micelles and leading to the blockage of the entrance to the enzymatic site. Detailed analysis of the simulations allow us to suggest a set of residues from the SDS binding site on HiC to probe as engineered mutations aimed at reducing SDS binding to HiC, thereby decreasing SDS inhibition of HiC.

DNA-encircled lipid bilayers.

Lipid bilayers and lipid-associated proteins play crucial roles in biology. As in vivo studies and manipulation are inherently difficult, membrane-mimetic systems are useful for the investigation of lipidic phases, lipid-protein interactions, membrane protein function and membrane structure in vitro. In this work, we describe a route to leverage the programmability of DNA nanotechnology and create DNA-encircled bilayers (DEBs). DEBs are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle, in which up to two alkyl chains per helical turn point to the inside of the toroidal DNA ring. When phospholipids are added, a bilayer is observed to self-assemble within the ring such that the alkyl chains of the oligonucleotides stabilize the hydrophobic rim of the bilayer to prevent formation of vesicles and support thermotropic lipid phase transitions. The DEBs are completely free of protein and can be synthesized from commercially available components using routine equipment. The diameter of DEBs can be varied in a predictable manner. The well-established toolbox from structural DNA nanotechnology, will ultimately enable the rational design of DEBs so that their size, shape or functionalization can be adapted to the specific needs of biophysical investigations of lipidic phases and the properties of membrane proteins embedded into DEB nanoparticle bilayers.

Insight into the molecular mechanism behind PEG-mediated stabilization of biofluid lipases.

Bioconjugates established between anionic polyethylene glycol (PEG) based polymers and cationic proteins have proven to be a promising strategy to engineer thermostable biocatalysts. However, the enzyme activity of these bioconjugates is very low and the mechanism of non-covalent PEG-stabilization is yet to be understood. This work presents experimental and molecular dynamics simulation studies, using lipase-polymer surfactant nanoconjugates from mesophile Rhizomucor miehei (RML), performed to evaluate the effect of PEG on enzyme stability and activity. Results demonstrated that the number of hydrogen bonds between the cationized RML and PEG chain correlates with enzyme thermostability. In addition, an increase of both the number of PEG-polymers units and cationization degree of the enzyme leads to a decrease of enzyme activity. Modelling with SAXS data of aqueous solutions of the biofluid lipases agrees with previous hypothesis that these enzymes contain a core constituted of folded protein confined by a shell of surfactants. Together results provide valuable insight into the mechanism of non-covalent PEG mediated protein stabilization relevant for engineering active and thermostable biofluids. Furthermore, the first biofluids RML with activity comparable to their cationized counterpart are presented.

Dimer Interface of the Human Serotonin Transporter and Effect of the Membrane Composition.

The oligomeric state of membrane proteins has recently emerged in many cases as having an effect on their function. However, the intrinsic dynamics of their spatial organization in cells and model systems makes it challenging to characterize. Here we use molecular dynamics (MD) simulations at multiple resolutions to determine the dimer conformation of the human serotonin transporter (hSERT). From self-assembly simulations we predict dimer candidates and subsequently quantify their relative strength. We use umbrella sampling (US) replica exchange MD simulations for which we present extensive analysis of their efficiency and improved sampling compared to regular US MD simulations. The data shows that the most stable hSERT dimer interface is symmetrical and involves transmembrane helix 12 (TM12), similar to the crystal structure of the bacterial homologue LeuT, but with a slightly different orientation. We also describe the supramolecular organization of hSERT from a 250 µs self-assembly simulation. Finally, the effects of the presence of phosphatidylinositol bisphosphate or cholesterol in the membrane model has been quantified for the TM12-TM12 predicted interface. Collectively, the presented data bring new insight to the area of protein and lipid interplay in biological membranes.

Cholesterol binding to a conserved site modulates the conformation, pharmacology, and transport kinetics of the human serotonin transporter.

The serotonin transporter (SERT) is important for reuptake of the neurotransmitter serotonin from the synaptic cleft and is also the target of most antidepressants. It has previously been shown that cholesterol in the membrane bilayer affects the conformation of SERT. Although recent crystal structures have identified several potential cholesterol-binding sites, it is unclear whether any of these potential cholesterol sites are occupied by cholesterol and functionally relevant. In the present study, we focus on the conserved cholesterol site 1 (CHOL1) located in a hydrophobic groove between TM1a, TM5, and TM7. By molecular dynamics simulations, we demonstrate a strong binding of cholesterol to CHOL1 in a membrane bilayer environment. In biochemical experiments, we find that cholesterol depletion induces a more inward-facing conformation favoring substrate analog binding. Consistent with this, we find that mutations in CHOL1 with a negative impact on cholesterol binding induce a more inward-facing conformation, and, vice versa, mutations with a positive impact on cholesterol binding induce a more outward-facing conformation. This shift in transporter conformation dictated by the ability to bind cholesterol in CHOL1 affects the apparent substrate affinity, maximum transport velocity, and turnover rates. Taken together, we show that occupation of CHOL1 by cholesterol is of major importance in the transporter conformational equilibrium, which in turn dictates ligand potency and serotonin transport activity. Based on our findings, we propose a mechanistic model that incorporates the role of cholesterol binding to CHOL1 in the function of SERT.

A direct interaction of cholesterol with the dopamine transporter prevents its out-to-inward transition.

Monoamine transporters (MATs) carry out neurotransmitter reuptake from the synaptic cleft, a key step in neurotransmission, which is targeted in the treatment of neurological disorders. Cholesterol (CHOL), a major component of the synaptic plasma membrane, has been shown to exhibit a modulatory effect on MATs. Recent crystal structures of the dopamine transporter (DAT) revealed the presence of two conserved CHOL-like molecules, suggesting a functional protein-CHOL direct interaction. Here, we present extensive atomistic molecular dynamics (MD) simulations of DAT in an outward-facing conformation. In the absence of bound CHOL, DAT undergoes structural changes reflecting early events of dopamine transport: transition to an inward-facing conformation. In contrast, in the presence of bound CHOL, these conformational changes are inhibited, seemingly by an immobilization of the intracellular interface of transmembrane helix 1a and 5 by CHOL. We also provide evidence, from coarse grain MD simulations that the CHOL sites observed in the DAT crystal structures are preserved in all human monoamine transporters (dopamine, serotonin and norepinephrine), suggesting that our findings might extend to the entire family.

Energetics Underlying Twist Polymorphisms in Amyloid Fibrils.

Amyloid fibrils are highly ordered protein aggregates associated with more than 40 human diseases. The exact conditions under which the fibrils are grown determine many types of reported fibril polymorphism, including different twist patterns. Twist-based polymorphs display unique mechanical properties in vitro, and the relevance of twist polymorphism in amyloid diseases has been suggested. We present transmission electron microscopy images of Aß42-derived (amyloid ß) fibrils, which are associated with Alzheimer's disease, demonstrating the presence of twist variability even within a single long fibril. To better understand the molecular underpinnings of twist polymorphism, we present a structural and thermodynamics analysis of molecular dynamics simulations of the twisting of ß-sheet protofilaments of a well-characterized cross-ß model: the GNNQQNY peptide from the yeast prion Sup35. The results show that a protofilament model of GNNQQNY is able to adopt twist angles from -11° on the left-hand side to +8° on the right-hand side in response to various external conditions, keeping an unchanged peptide structure. The potential of mean force (PMF) of this cross-ß structure upon twisting revealed that only ∼2kBT per peptide are needed to stabilize a straight conformation with respect to the left-handed free-energy minimum. The PMF also shows that the canonical structural core of ß-sheets, i.e., the hydrogen-bonded backbone ß-strands, favors the straight conformation. However, the concerted effects of the side chains contribute to twisting, which provides a rationale to correlate polypeptide sequence, environmental growth conditions and number of protofilaments in a fibril with twist polymorphisms.

Exchange pathways of plastoquinone and plastoquinol in the photosystem II complex.

Plastoquinone (PLQ) acts as an electron carrier between photosystem II (PSII) and the cytochrome b6f complex. To understand how PLQ enters and leaves PSII, here we show results of coarse grained molecular dynamics simulations of PSII embedded in the thylakoid membrane, covering a total simulation time of more than 0.5 ms. The long time scale allows the observation of many spontaneous entries of PLQ into PSII, and the unbinding of plastoquinol (PLQol) from the complex. In addition to the two known channels, we observe a third channel for PLQ/PLQol diffusion between the thylakoid membrane and the PLQ binding sites. Our simulations point to a promiscuous diffusion mechanism in which all three channels function as entry and exit channels. The exchange cavity serves as a PLQ reservoir. Our simulations provide a direct view on the exchange of electron carriers, a key step of the photosynthesis machinery.

Interplay of G Protein-Coupled Receptors with the Membrane: Insights from Supra-Atomic Coarse Grain Molecular Dynamics Simulations.

G protein-coupled receptors (GPCRs) are central to many fundamental cellular signaling pathways. They transduce signals from the outside to the inside of cells in physiological processes ranging from vision to immune response. It is extremely challenging to look at them individually using conventional experimental techniques. Recently, a pseudo atomistic molecular model has emerged as a valuable tool to access information on GPCRs, more specifically on their interactions with their environment in their native cell membrane and the consequences on their supramolecular organization. This approach uses the Martini coarse grain (CG) model to describe the receptors, lipids, and solvent in molecular dynamics (MD) simulations and in enough detail to allow conserving the chemical specificity of the different molecules. The elimination of unnecessary degrees of freedom has opened up large-scale simulations of the lipid-mediated supramolecular organization of GPCRs. Here, after introducing the Martini CGMD method, we review these studies carried out on various members of the GPCR family, including rhodopsin (visual receptor), opioid receptors, adrenergic receptors, adenosine receptors, dopamine receptor, and sphingosine 1-phosphate receptor. These studies have brought to light an interesting set of novel biophysical principles. The insights range from revealing localized and heterogeneous deformations of the membrane bilayer at the surface of the protein, specific interactions of lipid molecules with individual GPCRs, to the effect of the membrane matrix on global GPCR self-assembly. The review ends with an overview of the lessons learned from the use of the CGMD method, the biophysical-chemical findings on lipid-protein interplay.

Molecular Dynamics of Photosystem II Embedded in the Thylakoid Membrane.

Photosystem II (PSII) is one of the key protein complexes in photosynthesis. We introduce a coarse grained model of PSII and present the analysis of 60 µs molecular dynamics simulations of PSII in both monomeric and dimeric form, embedded in a thylakoid membrane model that reflects its native lipid composition. We describe in detail the setup of the protein complex and the many natural cofactors and characterize their mobility. Overall we find that the protein subunits and cofactors are more flexible toward the periphery of the complex as well as near the PLQ exchange cavity and at the dimer interface. Of all cofactors, ß-carotenes show the highest mobility. Some of the ß-carotenes diffuse in and out of the protein complex via the thylakoid membrane. In contrast with the PSII dimer, the monomeric form adopts a tilted conformation in the membrane, with strong interactions between the soluble PsbO subunit and the glycolipid headgroups. Interestingly, the tilted conformation causes buckling of the membrane. Together, our results provide an unprecedented view of PSII dynamics on a microsecond time scale. Our data may be used as basis for the interpretation of experimental data as well as for theoretical models describing exciton energy transfer.

An Amphotericin B Derivative Equally Potent to Amphotericin B and with Increased Safety.

Amphotericin B is the most potent antimycotic known to date. However due to its large collateral toxicity, its use, although long standing, had been limited. Many attempts have been made to produce derivatives with reduced collateral damage. The molecular mechanism of polyene has also been closely studied for this purpose and understanding it would contribute to the development of safe derivatives. Our study examined polyene action, including chemical synthesis, electrophysiology, pharmacology, toxicology and molecular dynamics. The results were used to support a novel Amphotericin B derivative with increased selectivity: L-histidine methyl ester of Amphotericin B. We found that this derivative has the same form of action as Amphotericin B, i.e. pore formation in the cell membrane. Its reduced dimerization in solution, when compared to Amphotericin B, is at least partially responsible for its increased selectivity. Here we also present the results of preclinical tests, which show that the derivative is just as potent as Amphotericin B and has increased safety.

Computational 'microscopy' of cellular membranes.

Computational 'microscopy' refers to the use of computational resources to simulate the dynamics of a molecular system. Tuned to cell membranes, this computational 'microscopy' technique is able to capture the interplay between lipids and proteins at a spatio-temporal resolution that is unmatched by other methods. Recent advances allow us to zoom out from individual atoms and molecules to supramolecular complexes and subcellular compartments that contain tens of millions of particles, and to capture the complexity of the crowded environment of real cell membranes. This Commentary gives an overview of the main concepts of computational 'microscopy' and describes the state-of-the-art methods used to model cell membrane processes. We illustrate the power of computational modelling approaches by providing a few in-depth examples of large-scale simulations that move up from molecular descriptions into the subcellular arena. We end with an outlook towards modelling a complete cell in silico.

Dry Martini, a coarse-grained force field for lipid membrane simulations with implicit solvent.

Coarse-grained (CG) models allow simulation of larger systems for longer times by decreasing the number of degrees of freedom compared with all-atom models. Here we introduce an implicit-solvent version of the popular CG Martini model, nicknamed "Dry" Martini. To account for the omitted solvent degrees of freedom, the nonbonded interaction matrix underlying the Martini force field was reparametrized. The Dry Martini force field reproduces relatively well a variety of lipid membrane properties such as area per lipid, bilayer thickness, bending modulus, and coexistence of liquid-ordered and disordered domains. Furthermore, we show that the new model can be applied to study membrane fusion and tether formation, with results similar to those of the standard Martini model. Membrane proteins can also be included, but less quantitative results are obtained. The absence of water in Dry Martini leads to a significant speedup for large systems, opening the way to the study of complex multicomponent membranes containing millions of lipids.

From light-harvesting to photoprotection: structural basis of the dynamic switch of the major antenna complex of plants (LHCII).

Light-Harvesting Complex II (LHCII) is largely responsible for light absorption and excitation energy transfer in plants in light-limiting conditions, while in high-light it participates in photoprotection. It is generally believed that LHCII can change its function by switching between different conformations. However, the underlying molecular picture has not been elucidated yet. The available crystal structures represent the quenched form of the complex, while solubilized LHCII has the properties of the unquenched state. To determine the structural changes involved in the switch and to identify potential quenching sites, we have explored the structural dynamics of LHCII, by performing a series of microsecond Molecular Dynamics simulations. We show that LHCII in the membrane differs substantially from the crystal and has the signatures that were experimentally associated with the light-harvesting state. Local conformational changes at the N-terminus and at the xanthophyll neoxanthin are found to strongly correlate with changes in the interactions energies of two putative quenching sites. In particular conformational disorder is observed at the terminal emitter resulting in large variations of the excitonic coupling strength of this chlorophyll pair. Our results strongly support the hypothesis that light-harvesting regulation in LHCII is coupled with structural changes.

Atomistic and Coarse Grain Topologies for the Cofactors Associated with the Photosystem II Core Complex.

Electron transfers within and between protein complexes are core processes of the electron transport chains occurring in thylakoid (chloroplast), mitochondrial, and bacterial membranes. These electron transfers involve a number of cofactors. Here we describe the derivation of molecular mechanics parameters for the cofactors associated with the function of the photosystem II core complex: plastoquinone, plastoquinol, heme b, chlorophyll A, pheophytin, and ß-carotene. Parameters were also obtained for ubiquinol and ubiquinone, related cofactors involved in the respiratory chain. Parameters were derived at both atomistic and coarse grain (CG) resolutions, compatible with the building blocks of the GROMOS united-atom and Martini CG force fields, respectively. Structural and thermodynamic properties of the cofactors were compared to experimental values when available. The topologies were further tested in molecular dynamics simulations of the cofactors in their physiological environment, e.g., either in a lipid membrane environment or in complex with the heme binding protein bacterioferritin.

The power of coarse graining in biomolecular simulations.

Computational modeling of biological systems is challenging because of the multitude of spatial and temporal scales involved. Replacing atomistic detail with lower resolution, coarse grained (CG), beads has opened the way to simulate large-scale biomolecular processes on time scales inaccessible to all-atom models. We provide an overview of some of the more popular CG models used in biomolecular applications to date, focusing on models that retain chemical specificity. A few state-of-the-art examples of protein folding, membrane protein gating and self-assembly, DNA hybridization, and modeling of carbohydrate fibers are used to illustrate the power and diversity of current CG modeling.

Lipid organization of the plasma membrane.

The detailed organization of cellular membranes remains rather elusive. Based on large-scale molecular dynamics simulations, we provide a high-resolution view of the lipid organization of a plasma membrane at an unprecedented level of complexity. Our plasma membrane model consists of 63 different lipid species, combining 14 types of headgroups and 11 types of tails asymmetrically distributed across the two leaflets, closely mimicking an idealized mammalian plasma membrane. We observe an enrichment of cholesterol in the outer leaflet and a general non-ideal lateral mixing of the different lipid species. Transient domains with liquid-ordered character form and disappear on the microsecond time scale. These domains are coupled across the two membrane leaflets. In the outer leaflet, distinct nanodomains consisting of gangliosides are observed. Phosphoinositides show preferential clustering in the inner leaflet. Our data provide a key view on the lateral organization of lipids in one of life's fundamental structures, the cell membrane.