Psychedelics promote plasticity by directly binding to BDNF receptor TrkB.

Psychedelics produce fast and persistent antidepressant effects and induce neuroplasticity resembling the effects of clinically approved antidepressants. We recently reported that pharmacologically diverse antidepressants, including fluoxetine and ketamine, act by binding to TrkB, the receptor for BDNF. Here we show that lysergic acid diethylamide (LSD) and psilocin directly bind to TrkB with affinities 1,000-fold higher than those for other antidepressants, and that psychedelics and antidepressants bind to distinct but partially overlapping sites within the transmembrane domain of TrkB dimers. The effects of psychedelics on neurotrophic signaling, plasticity and antidepressant-like behavior in mice depend on TrkB binding and promotion of endogenous BDNF signaling but are independent of serotonin 2A receptor (5-HT2A) activation, whereas LSD-induced head twitching is dependent on 5-HT2A and independent of TrkB binding. Our data confirm TrkB as a common primary target for antidepressants and suggest that high-affinity TrkB positive allosteric modulators lacking 5-HT2A activity may retain the antidepressant potential of psychedelics without hallucinogenic effects.

Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties.

The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades, several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis versus trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

Maturation of the SARS-CoV-2 virus is regulated by dimerization of its main protease.

SARS-CoV-2 main protease (Mpro) involved in COVID-19 is required for maturation of the virus and infection of host cells. The key question is how to block the activity of Mpro. By combining atomistic simulations with machine learning, we found that the enzyme regulates its own activity by a collective allosteric mechanism that involves dimerization and binding of a single substrate. At the core of the collective mechanism is the coupling between the catalytic site residues, H41 and C145, which direct the activity of Mpro dimer, and two salt bridges formed between R4 and E290 at the dimer interface. If these salt bridges are mutated, the activity of Mpro is blocked. The results suggest that dimerization of main proteases is a general mechanism to foster coronavirus proliferation, and propose a robust drug-based strategy that does not depend on the frequently mutating spike proteins at the viral envelope used to develop vaccines.

Examining the Effect of Charged Lipids on Mitochondrial Outer Membrane Dynamics Using Atomistic Simulations.

The outer mitochondrial membrane (OMM) is involved in multiple cellular functions such as apoptosis, inflammation and signaling via its membrane-associated and -embedded proteins. Despite the central role of the OMM in these vital phenomena, the structure and dynamics of the membrane have regularly been investigated in silico using simple two-component models. Accordingly, the aim was to generate the realistic multi-component model of the OMM and inspect its properties using atomistic molecular dynamics (MD) simulations. All major lipid components, phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), were included in the probed OMM models. Because increased levels of anionic PS lipids have potential effects on schizophrenia and, more specifically, on monoamine oxidase B enzyme activity, the effect of varying the PS concentration was explored. The MD simulations indicate that the complex membrane lipid composition (MLC) behavior is notably different from the two-component PC-PE model. The MLC changes caused relatively minor effects on the membrane structural properties such as membrane thickness or area per lipid; however, notable effects could be seen with the dynamical parameters at the water-membrane interface. Increase of PS levels appears to slow down lateral diffusion of all lipids and, in general, the presence of anionic lipids reduced hydration and slowed down the PE headgroup rotation. In addition, sodium ions could neutralize the membrane surface, when PI was the main anionic component; however, a similar effect was not seen for high PS levels. Based on these results, it is advisable for future studies on the OMM and its protein or ligand partners, especially when wanting to replicate the correct properties on the water-membrane interface, to use models that are sufficiently complex, containing anionic lipid types, PI in particular.

Mechanistic Understanding from Molecular Dynamics in Pharmaceutical Research 2: Lipid Membrane in Drug Design.

We review the use of molecular dynamics (MD) simulation as a drug design tool in the context of the role that the lipid membrane can play in drug action, i.e., the interaction between candidate drug molecules and lipid membranes. In the standard "lock and key" paradigm, only the interaction between the drug and a specific active site of a specific protein is considered; the environment in which the drug acts is, from a biophysical perspective, far more complex than this. The possible mechanisms though which a drug can be designed to tinker with physiological processes are significantly broader than merely fitting to a single active site of a single protein. In this paper, we focus on the role of the lipid membrane, arguably the most important element outside the proteins themselves, as a case study. We discuss work that has been carried out, using MD simulation, concerning the transfection of drugs through membranes that act as biological barriers in the path of the drugs, the behavior of drug molecules within membranes, how their collective behavior can affect the structure and properties of the membrane and, finally, the role lipid membranes, to which the vast majority of drug target proteins are associated, can play in mediating the interaction between drug and target protein. This review paper is the second in a two-part series covering MD simulation as a tool in pharmaceutical research; both are designed as pedagogical review papers aimed at both pharmaceutical scientists interested in exploring how the tool of MD simulation can be applied to their research and computational scientists interested in exploring the possibility of a pharmaceutical context for their research.

Mechanistic Insight into How PEGylation Reduces the Efficacy of pH-Sensitive Liposomes from Molecular Dynamics Simulations.

Liposome-based drug delivery systems composed of DOPE stabilized with cholesteryl hemisuccinate (CHMS) have been proposed as a drug delivery mechanism with pH-triggered release as the anionic form (CHSa) is protonated (CHS) at reduced pH; PEGylation is known to decrease this pH sensitivity. In this manuscript, we set out to use molecular dynamics (MD) simulations with a model with all-atom resolution to provide insight into why incorporation of poly(ethyleneglycol) (PEG) into DOPE-CHMS liposomes reduces their pH sensitivity; we also address two additional questions: (1) How CHSa stabilizes DOPE bilayers into a lamellar conformation at a physiological pH of 7.4? and (2) how the change from CHSa to CHS at acidic pH triggers the destabilization of DOPE bilayers? We found that (A) CHSa stabilizes the DOPE lipid membrane by increasing the hydrophilicity of the bilayer surface, (B) when CHSa changes to CHS by pH reduction, DOPE bilayers are destabilized due to a reduction in bilayer hydrophilicity and a reduction in the area per lipid, and (C) PEG stabilizes DOPE bilayers into the lamellar phase, thus reducing the pH sensitivity of the liposomes by increasing the area per lipid through penetration into the bilayer, which is our main focus.

Can di-4-ANEPPDHQ reveal the structural differences between nanodiscs and liposomes?

The potential-sensitive di-4-ANEPPDHQ dye is presently gaining popularity in structural studies of the lipid bilayer. Within the bilayer, dye environmental sensitivity originates from the excitation induced charge redistribution and is usually attributed to solvent relaxation. Here, di-4-ANEPPDHQ is utilized to compare the structure of neutral and negatively charged lipid bilayers between two model systems: the nanodiscs and the liposomes. Using the well-established approach of measuring solvatochromic shifts of the steady-state spectra to study the bilayer structural changes has proved insufficient in this case. By applying an in-depth analysis of time-resolved fluorescence decays and emission spectra, we distinguished and characterized two and three distinct emissive di-4-ANEPPDHQ species in the liposomes and the nanodiscs, respectively. These emissive species were ascribed to the dual emission of the dye rather than to solvent relaxation. An additional, long-lived component present in the nanodiscs was associated with a unique domain of high order, postulated recently. Our results reveal that the di-4-ANEPPDHQ steady-state fluorescence should be interpreted with caution. With the experimental approach presented here, the di-4-ANEPPDHQ sensitivity was improved. We confirmed that the bilayer structure is, indeed, altered in the nanodiscs. Moreover, molecular dynamic simulations showed a distribution of the probe in the nanodiscs plane, which is sensitive to lipid composition. In POPC nanodiscs, probe frequently interacts with MSP, while in POPC-POPG nanodiscs, such interactions are rare. We did not observe, however, any impact of those interactions on the probe fluorescence.

Cholesterol-recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: The case of TRKB.

Cholesterol is an essential constituent of cell membranes. The discovery of cholesterol-recognition amino acid consensus (CRAC) motif in proteins indicated a putative direct, non-covalent interaction between cholesterol and proteins. In the present study, we evaluated the presence of a CRAC motif and its inverted version (CARC) in the transmembrane region (TMR) of the tyrosine kinase receptor family (RTK) in several species using in silico methods. CRAC motifs were found across all species analyzed, while CARC was found only in vertebrates. The tropomyosin-related kinase B (TRKB), a member of the RTK family, through interaction with its endogenous ligand brain-derived neurotrophic factor (BDNF) is a core participant in the neuronal plasticity process and exhibits a CARC motif in its TMR. Upon identifying the conserved CARC motif in the TRKB, we performed molecular dynamics simulations of the mouse TRKB.TMR. The simulations indicated that cholesterol interaction with the TRKB CARC motif occurs mainly at the central Y433 residue. Our binding assay suggested a bell-shaped effect of cholesterol on BDNF interaction with TRKB receptors, and our results suggest that CARC/CRAC motifs may play a role in the function of the RTK family TMR.

Antidepressant drugs act by directly binding to TRKB neurotrophin receptors.

It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo. We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.

Mechanistic Understanding From Molecular Dynamics Simulation in Pharmaceutical Research 1: Drug Delivery.

In this review, we outline the growing role that molecular dynamics simulation is able to play as a design tool in drug delivery. We cover both the pharmaceutical and computational backgrounds, in a pedagogical fashion, as this review is designed to be equally accessible to pharmaceutical researchers interested in what this new computational tool is capable of and experts in molecular modeling who wish to pursue pharmaceutical applications as a context for their research. The field has become too broad for us to concisely describe all work that has been carried out; many comprehensive reviews on subtopics of this area are cited. We discuss the insight molecular dynamics modeling has provided in dissolution and solubility, however, the majority of the discussion is focused on nanomedicine: the development of nanoscale drug delivery vehicles. Here we focus on three areas where molecular dynamics modeling has had a particularly strong impact: (1) behavior in the bloodstream and protective polymer corona, (2) Drug loading and controlled release, and (3) Nanoparticle interaction with both model and biological membranes. We conclude with some thoughts on the role that molecular dynamics simulation can grow to play in the development of new drug delivery systems.

The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering.

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the ß integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+ Here we show that kindlin-1 can replace Mn2+ to mediate ß3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ß3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and ß3-integrins, in order to activate the ß3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ß3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer.This article has an associated First Person interview with the first author of the paper.

Rigorous Computational Study Reveals What Docking Overlooks: Double Trouble from Membrane Association in Protein Kinase C Modulators.

Increasing protein kinase C (PKC) activity is of potential therapeutic value. Its activation involves an interaction between the C1 domain and diacylglycerol (DAG) at intracellular membrane surfaces; DAG mimetics hold promise as new drugs. We previously developed the isophthalate derivative HMI-1a3, an effective but highly lipophilic (clogP = 6.46) DAG mimetic. Although a less lipophilic pyrimidine analog, PYR-1gP (clogP = 3.30), gave positive results in computational docking, it unexpectedly presented greatly diminished binding to PKC in vitro. Through more rigorous computational molecular modeling, we reveal that, unlike HMI-1a3, PYR-1gP forms an intramolecular hydrogen bond, which both obstructs binding and reorients PYR-1gP in the membrane in a fashion that prevents it from correctly accessing the PKC C1 domain. Our results highlight the great value of molecular dynamics simulations as a key component for the drug design process of ligands targeting weakly membrane-associated proteins, where simulation in the relevant membrane environment is crucial for obtaining biologically applicable results.

Cooperative Effects of an Antifungal Moiety and DMSO on Pore Formation over Lipid Membranes Revealed by Free Energy Calculations.

Itraconazole is a triazole drug widely used in the treatment of fungal infections, and it is in clinical trials for treatment of several cancers. However, the drug suffers from poor solubility, while experiments have shown that itraconazole delivery in liposome nanocarriers improves both circulation half-life and tissue distribution. The drug release mechanism from the nanocarrier is still unknown, and it depends on several factors including membrane stability against defect formation. In this work, we used molecular dynamics simulations and potential of mean force (PMF) calculations to quantify the influence of itraconazole on pore formation over lipid membranes, and we compared the effect by itraconazole with a pore-stabilizing effect by the organic solvent dimethyl sulfoxide (DMSO). According to the PMFs, both itraconazole and DMSO greatly reduce the free energy of pore formation, by up to ∼20 kJ mol-1. However, whereas large concentrations of itraconazole of 8 mol % (relative to lipid) were required, only small concentrations of a few mole % DMSO (relative to water) were sufficient to stabilize pores. In addition, itraconazole and DMSO facilitate pore formation by different mechanisms. Whereas itraconazole predominantly aids the formation of a partial defect with a locally thinned membrane, DMSO mainly stabilizes a transmembrane water needle by shielding it from the hydrophobic core. Notably, the two distinct mechanisms act cooperatively upon adding both itraconazole and DMSO to the membrane, as revealed by an additional reduction of the pore free energy. Overall, our simulations reveal molecular mechanisms and free energies of membrane pore formation by small molecules. We suggest that the stabilization of a locally thinned membrane as well as the shielding of a transmembrane water needle from the hydrophobic membrane core may be a general mechanism by which amphiphilic molecules facilitate pore formation over lipid membranes at sufficient concentrations.

Tail-Oxidized Cholesterol Enhances Membrane Permeability for Small Solutes.

Cholesterol renders mammalian cell membranes more compact by reducing the amount of voids in the membrane structure. Because of this, cholesterol is known to regulate the ability of cell membranes to prevent the permeation of water and water-soluble molecules through the membranes. Meanwhile, it is also known that even seemingly tiny modifications in the chemical structure of cholesterol can lead to notable changes in membrane properties. The question is, how significantly do these small changes in cholesterol structure affect the permeability barrier function of cell membranes? In this work, we applied fluorescence methods as well as atomistic molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7ß-hydroxycholesterol (7ß-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that the oxidation of the cholesterol tail has only a marginal effect on the structure of a lipid bilayer; however, oxidation was found to disturb membrane dynamics by introducing a mechanism that allows sterol molecules to move rapidly back and forth across the membrane-bobbing. Herein, we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid probes in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to the conclusion that the presence of 27-OH-chol increases membrane permeability to the dithionite anion. Atomistic molecular dynamics simulations demonstrated that 27-OH-chol also facilitates water transport across the membrane. The results support the view that oxysterol bobbing gives rise to successive perturbations to the hydrophobic core of the membrane, and these perturbations promote the permeation of water and small water-soluble molecules through a lipid bilayer. The observed impairment of permeability can have important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7ß-OH-chol which represents ring-oxidized sterols.

Complexity of seemingly simple lipid nanodiscs.

Lipid nanodiscs are macromolecular assemblies, where a scaffold protein is wrapped around a nanosized disc of a lipid bilayer, thus protecting the hydrocarbon chains at the disc edges from unfavorable interactions with water. These nanostructures have numerous applications in, e.g., nanotechnology and pharmaceutics, and in investigations of membrane proteins. Here, we present results based on atomistic molecular dynamics simulations combined with electron paramagnetic spectroscopy measurements on the structure and dynamics of lipids in single-component nanodiscs. Our data highlight the existence of three distinctly different lipid fractions: central lipids residing in the center of a nanodisc, boundary lipids in direct contact with a scaffold protein, and intermediate lipids between these two regions. The central lipids are highly ordered and characterized by slow diffusion. In this part of the nanodisc, the membrane is the thickest and characterized by a gel-like or liquid-ordered phase, having features common to cholesterol-rich membranes. The boundary lipids in direct contact with the scaffold protein turned out to be less ordered and characterized by faster diffusion, and they remained in the liquid-disordered phase even at temperatures that were somewhat below the main phase transition temperature (Tm). The enthalpies associated with the central-boundary and central-intermediate transitions were similar to those observed for lipids going through the main phase transition. Overall, the study reveals lipid nanodiscs to be characterized by a complex internal structure, which is expected to influence membrane proteins placed in nanodiscs.

Membrane-Dependent Binding and Entry Mechanism of Dopamine into Its Receptor.

Synaptic neurotransmission has recently been proposed to function via either a membrane-independent or a membrane-dependent mechanism, depending on the neurotransmitter type. In the membrane-dependent mechanism, amphipathic neurotransmitters first partition to the lipid headgroup region and then diffuse along the membrane plane to their membrane-buried receptors. However, to date, this mechanism has not been demonstrated for any neurotransmitter-receptor complex. Here, we combined isothermal calorimetry measurements with a diverse set of molecular dynamics simulation methods to investigate the partitioning of an amphipathic neurotransmitter (dopamine) and the mechanism of its entry into the ligand-binding site. Our results show that the binding of dopamine to its receptor is consistent with the membrane-dependent binding and entry mechanism. Both experimental and simulation results showed that dopamine favors binding to lipid membranes especially in the headgroup region. Moreover, our simulations revealed a ligand-entry pathway from the membrane to the binding site. This pathway passes through a lateral gate between transmembrane alpha-helices 5 and 6 on the membrane-facing side of the protein. All in all, our results demonstrate that dopamine binds to its receptor by a membrane-dependent mechanism, and this is complemented by the more traditional binding mechanism directly through the aqueous phase. The results suggest that the membrane-dependent mechanism is common in other synaptic receptors, too.

Cholesterol Reduces Partitioning of Antifungal Drug Itraconazole into Lipid Bilayers.

Cholesterol plays a crucial role in modulating the physicochemical properties of biomembranes, both increasing mechanical strength and decreasing permeability. Cholesterol is also a common component of vesicle-based delivery systems, including liposome-based drug delivery systems (LDSs). However, its effect on the partitioning of drug molecules to lipid membranes is very poorly recognized. Herein, we performed a combined experimental/computational study of the potential for the use of the LDS formulation for the delivery of the antifungal drug itraconazole (ITZ). We consider the addition of cholesterol to the lipid membrane. Since ITZ is only weakly soluble in water, its bioavailability is limited. Use of an LDS has thus been proposed. We studied lipid membranes composed of cholesterol, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and ITZ using a combination of computational molecular dynamics (MD) simulations of lipid bilayers and Brewster angle microscopy (BAM) experiments of monolayers. Both experimental and computational results show separation of cholesterol and ITZ. Cholesterol has a strong preference to orient parallel to the bilayer normal. However, ITZ, a long and relatively rigid molecule with weakly hydrophilic groups along the backbone, predominantly locates below the interface between the hydrocarbon chain region and the polar region of the membrane, with its backbone oriented parallel to the membrane surface; the orthogonal orientation in the membrane could be the cause of the observed separation. In addition, fluorescence measurements demonstrated that the affinity of ITZ for the lipid membrane is decreased by the presence of cholesterol, which is thus probably not a suitable formulation component of an LDS designed for ITZ delivery.

Syndecan-4 tunes cell mechanics by activating the kindlin-integrin-RhoA pathway.

Extensive research over the past decades has identified integrins to be the primary transmembrane receptors that enable cells to respond to external mechanical cues. We reveal here a mechanism whereby syndecan-4 tunes cell mechanics in response to localized tension via a coordinated mechanochemical signalling response that involves activation of two other receptors: epidermal growth factor receptor and ß1 integrin. Tension on syndecan-4 induces cell-wide activation of the kindlin-2/ß1 integrin/RhoA axis in a PI3K-dependent manner. Furthermore, syndecan-4-mediated tension at the cell-extracellular matrix interface is required for yes-associated protein activation. Extracellular tension on syndecan-4 triggers a conformational change in the cytoplasmic domain, the variable region of which is indispensable for the mechanical adaptation to force, facilitating the assembly of a syndecan-4/α-actinin/F-actin molecular scaffold at the bead adhesion. This mechanotransduction pathway for syndecan-4 should have immediate implications for the broader field of mechanobiology.

A Perspective: Active Role of Lipids in Neurotransmitter Dynamics.

Synaptic neurotransmission is generally considered as a function of membrane-embedded receptors and ion channels in response to the neurotransmitter (NT) release and binding. This perspective aims to widen the protein-centric view by including another vital component-the synaptic membrane-in the discussion. A vast set of atomistic molecular dynamics simulations and biophysical experiments indicate that NTs are divided into membrane-binding and membrane-nonbinding categories. The binary choice takes place at the water-membrane interface and follows closely the positioning of the receptors' binding sites in relation to the membrane. Accordingly, when a lipophilic NT is on route to a membrane-buried binding site, it adheres on the membrane and, then, travels along its plane towards the receptor. In contrast, lipophobic NTs, which are destined to bind into receptors with extracellular binding sites, prefer the water phase. This membrane-based sorting splits the neurotransmission into membrane-independent and membrane-dependent mechanisms and should make the NT binding into the receptors more efficient than random diffusion would allow. The potential implications and notable exceptions to the mechanisms are discussed here. Importantly, maintaining specific membrane lipid compositions (MLCs) at the synapses, especially regarding anionic lipids, affect the level of NT-membrane association. These effects provide a plausible link between the MLC imbalances and neurological diseases such as depression or Parkinson's disease. Moreover, the membrane plays a vital role in other phases of the NT life cycle, including storage and release from the synaptic vesicles, transport from the synaptic cleft, as well as their synthesis and degradation.

Cholesteryl Hemisuccinate Is Not a Good Replacement for Cholesterol in Lipid Nanodiscs.

Nanodiscs are suitable tools for studies of membrane proteins (MPs) due to their ability to mimic native biological membranes, and several MP structures are solved in nanodiscs. Among the various cell membrane components, cholesterol (CHL) is known to regulate protein function and its concentration can reach up to 50 mol %. However, studies comprising cholesterol are challenging due to its hydrophobic nature, hence, nanodiscs with only a low cholesterol concentration have been studied. To overcome the problem, cholesterol analogs with high solubility in polar solutions are often used, and one of them is cholesteryl hemisuccinate (CHS). Nevertheless, in molecular dynamics (MD) simulation, this is not an obstacle. In this study, we performed MD simulations of nanodiscs containing neutral phosphatidylcholine (POPC) lipids, negatively charged phosphatidylglycerol (POPG) lipids, CHL, or negatively charged cholesterol analog, CHS. Our simulations show that CHS increases the order of lipids in nanodiscs; the effect is, however, weaker than CHL and even smaller in nanodiscs. Furthermore, CHS gathered around scaffold proteins while cholesterol was uniformly distributed in the nanodiscs. Thus, nanodiscs with CHS are heterogeneous and not equivalent to nanodiscs with CHL. Finally, we also observed the increased concentration of POPG near the scaffold proteins, driven by electrostatic interactions. The MD results are experimentally validated using electron paramagnetic resonance spectroscopy. These results show that nanodiscs are, in fact, complex structures not easily comparable with planar lipid bilayers.