The cell membrane must balance mechanical stability with fluidity to function as both a barrier and an organizational platform. Key to this balance is the ordering of hydrocarbon chains and the packing of lipids. Many eukaryotes synthesize sterols, which are uniquely capable of modulating the lipid order to decouple membrane stability from fluidity. Ancient sterol analogs known as hopanoids are found in many bacteria and proposed as ancestral ordering lipids. The juxtaposition of sterols and hopanoids in extant organisms prompts us to ask why both pathways persist, especially in light of their convergent ability to order lipids. In this work, simulations, monolayer experiments, and cellular assays show that hopanoids and sterols order unsaturated phospholipids differently based on the position of double bonds in the phospholipid acyl chain. We find that cholesterol and diplopterol's methyl group distributions lead to distinct effects on unsaturated lipids. In Mesoplasma florum, diplopterol's constrained ordering capacity reduces membrane resistance to osmotic stress, unlike cholesterol. These findings suggest that cholesterol's broader lipid-ordering ability may have facilitated the exploration of a more diverse lipidomic landscape in eukaryotic membranes.

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A 2A adenosine receptor (A 2A AR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A 2A AR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

The cell membrane acts as a responsive interface between the cell and its surroundings. Ultimately, the diverse panel of lipids comprising the lipidome are employed to tune membrane biophysical properties for optimal function. For eukaryotes, a family of amphiphiles called sterols are crucial due to their unique capacity to modulate the order of membrane lipids. Bacteria typically lack sterols, however, some can synthesize a family of sterol-analogues called hopanoids. Hopanoids are recognized as bacterial analogs of sterols due to their chemical and biophysical similarities. Notably, hopanoids are proposed as evolutionary sterol precursors since they have been found in ancient sediments and their biosynthesis could have proceeded prior to the oxygenation of Earth’s surface. While hopanoids and sterols can both impart order to saturated phospholipids, this interaction changes vastly with the presence of a double bond in the phospholipid’s acyl chain. Here, we present a study examining how the unsaturation position along the acyl chain influences the ordering effect of sterols (cholesterol) and hopanoids (diplopterol). We found that diplopterol and cholesterol exhibit different ordering effects on unsaturated lipids, depending on the relative positions of the double bond and the methyl groups of cholesterol/diplopterol. Moreover, in the bacterium Mesoplasma florum, diplopterol’s interplay with unsaturated lipid isomers modulates bacterial membrane robustness. These results reveal how subtle changes in lipid structure can influence the membrane’s collective properties and introduces double bond position as a modifiable lipidomic feature that cells can employ to fine-tune their membrane for adaptation to environmental change.

19F NMR of A2AAR in nanodiscs of defined composition indicates that negative charge modulates activation of the receptor. The fully active state is obtained either when bound to G-protein, or without G-protein but in a membrane environment containing negatively charged headgroups. Mutagenesis indicates that a trio of residues on the intracellular ends of TM6 and TM7 are responsible for the lipid-dependent activity. In order to determine the mechanism, a series of molecular dynamics simulations were performed. Comparison of simulations of the inactive and the active, G-protein bound state shows that a glutamic acid on the helix of the G-protein coordinates interactions between these three residues. This suggested a hypothesis in which a negatively charged headgroup can stand-in for the glutamic acid side chain, coordinating the ends of TM6 and TM7 and preconditioning the receptor for G-protein coupling. To test this idea, simulations of the fully active state were performed with and without phosphatidyl serine headgroups, but without the G-protein. Consistent with our hypothesis, the TM6 -- TM7 interactions are similar to the G-protein coupled state in the presence of PS, but similar to the inactive state in the absence of PS. Moreover, the interaction of a PS headgroup with a key residue on TM6 recapitulates the same interaction with the glutamic acid, but only when the receptor is in the active state.

Lipid-protein interactions are critical for integral membrane protein function, where lipids can play the role of both solvent and ligand. Because resolving the lipids that solvate a membrane protein is experimentally challenging, simulations have come to play a prominent role in linking lipid solvation and membrane protein function. However, converging the lipid distribution around a membrane protein can require surprisingly long simulations, especially in cholesterol-rich mixtures where lipid diffusion and exchange at the surface of the protein can be quite slow.

G protein-coupled receptors (GPCRs) are sensory proteins that comprise the largest class of “druggable” targets. Earlier NMR studies demonstrated that GPCRs exist in a function-related equilibrium of simultaneously populated conformers, the relative populations of which are determined by the efficacy of bound drugs. We now present NMR data that show membrane phospholipids and sterols also control this equilibrium with a magnitude comparable to or greater than the influence of drug efficacy. Using NMR spectroscopy in aqueous solutions, we investigated the structural basis for the regulation of GPCR activity by phospholipids in nanodiscs containing the human A2A adenosine receptor (A2AAR), a representative class A GPCR. Nanodiscs containing mixtures of Zwitterionic and charged phospholipids yielded homogeneous preparations of fully functional receptor. We systematically investigated the influence of lipid composition over a wide range of mixtures of charged and Zwitterionic phospholipids using 19F NMR with conformationally-sensitive probes located at the A2AAR intracellular surface. Across all lipid compositions, A2AAR complexes with antagonists showed little response to variation in lipid composition. In striking contrast to this, A2AAR complexes with agonists responded dramatically to changes in the relative amounts of anionic phospholipids. A structural basis for these observations was investigated by molecular dynamics (MD) simulations and NMR spectroscopy with A2AAR variants containing replacements of charged residues with neutral amino acids. In 19F NMR experiments, replacement of charged residues in helix VI resulted in reduction of an active state population. In contrast, replacement of charged residues in helix VII showed an opposing effect. These results indicate that lipid composition should be carefully selected in all studies employing nanodiscs, and our data also provide a potential molecular mechanism for how changes in membrane composition can directly impact the efficacy of GPCR drugs.

Endogenous phospholipids are widely recognized to regulate membrane protein structure and activity through specific protein-lipid interactions and by altering membrane bulk chemical and physical properties. Protein-lipid interactions are important regulators of G protein-coupled receptors (GPCRs), sensory membrane proteins that control many physiological processes and targets of over one-third of FDA-approved drugs. However, molecular mechanisms by which phospholipids regulate GPCRs are not well understood.

Sphingolipids synthesis was thought to be rare in Gram-negative bacteria, previously only found in a handful of taxa. We recently discovered ceramides in Caulobacter crescentus and demonstrated that these lipids play an important role in antibiotic and phage sensitivity. However, the mechanism by which ceramides affect resistance to antimicrobials, as well as their effects on the integrity of the cell membrane are not yet clear. In this study, a coarse-grained molecular dynamics simulation of a prototypical bacterial outer membrane is used to observe changes in the conformation of the outer membrane lipids in the presence of ceramides. The outer membrane of a Gram-negative bacteria is asymmetric with an outer leaflet dominated by lipopolysaccharide (LPS). LPS is composed of three domains that extend from the outer membrane: a membrane-embedded lipid A, the attached core polysaccharide chain, and the O-antigen polysaccharide chain. Rough LPS consists of lipid A and core oligosaccharides only, while smooth LPS contains lipid A, core sugars, and O-antigen chains. Membranes were simulated with a one to one ratio of rough to smooth LPS, with ceramide concentrations ranging from ten to forty percent of total lipids. In order to understand the role of ceramide in outer membrane structure and function, this study considers their effects on the flexibility of O-antigen as well as the clustering and packing of LPS and membrane lipids.

The nicotinic acetylcholine receptor (nAChR) and other pentameric ligand-gated ion channels are native to neuronal membranes with an unusual lipid composition. While it is well-established that these receptors can be significantly modulated by lipids, the underlying mechanisms have been primarily studied in model membranes with few lipid species. Here, we use coarse-grained molecular dynamics simulation to probe specific binding of lipids in a complex quasi-neuronal membrane. We ran a total of 50 μs of simulations of a single nAChR in a membrane composed of 36 species of lipids. Competition between multiple lipid species produces a complex distribution. We find that overall, cholesterol selects for concave inter-subunit sites and polyunsaturated fatty acids select for convex M4 sites, while monounsaturated and saturated lipids are unenriched in the nAChR boundary. We propose the "density-threshold affinity" as a metric calculated from continuous density distributions, which reduces to a standard affinity in two-state binding. We find that the density-threshold affinity for M4 weakens with chain rigidity, which suggests that flexible chains may help relax packing defects caused by the conical protein shape. For any site, PE headgroups have the strongest affinity of all phospholipid headgroups, but anionic lipids still yield moderately high affinities for the M4 sites as expected. We observe cooperative effects between anionic headgroups and saturated chains at the M4 site in the inner leaflet. We also analyze affinities for individual anionic headgroups. When combined, these insights may reconcile several apparently contradictory experiments on the role of anionic phospholipids in modulating nAChR.

The nicotinic acetylcholine receptor (nAChR) is a highly lipid-sensitive neurotransmitter receptor and pentameric ligand gated ion channel. We previously used coarse-grained molecular dynamics (CG-MD) simulations to investigate boundary lipids of a single nAChR in model domain-forming membranes, with neutral head groups and symmetric leaflets. We observed that nAChRs partition into a liquid-disordered domain if such a domain exists, and also quantified specific boundary interactions with both cholesterol and polyunsaturated fatty acids. The simulated systems were distinct from native membranes in several critical ways: they did not include charged head-groups, asymmetric leaflets, multiple proteins, or the heteroacidic lipids that reduce domain formation. The current research focuses on overcoming these limitations by introducing multiple proteins into quasi-native membranes, including membranes based on rat synapses. We characterize the effects of increased membrane complexity on boundary lipid diversity, the role of leaflet asymmetry in determining boundary lipid asymmetry, and the role of head-group charge vs. chain unsaturation in determining affinity for the nAChR TMD. We find that the presence of domains reduces the concentration of nAChRs required for dimerization, but that formation of dimers and higher-order oligomers does not require domain formation. Finally, we investigate the effects of oligomerization on deformations of the surrounding membrane and associated lipid sorting.

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, a mutation that decreases ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.

The nicotinic acetylcholine receptor (nAChR) is an excitatory neurotransmitter receptor that mediates muscle functioning by forming nAChR-associated, lattice networks. At the neuromuscular junction (NMJ), synaptic and intracellular proteins, notably Agrin, MusK, and rapsyn, ultimately stabilize these highly dense networks. Experimental evidence suggests that cholesterol-rich domains, known as lipid rafts, facilitate signaling among Agrin-Musk and rapsyn, and their presence is essential for healthy nAChR clustering. In spite of their importance, the structural and functional mechanisms of lipid domains are currently unknown. Alongside cholesterol, the omega-3 fatty acid, Docosahexaenoic acid (DHA), is prevalent at the NMJ, correlates with domain formation, and strongly promotes neuronal health. In the present study, molecular dynamics simulations were used to explore the role of DHA on nAChR clustering in the presence and absence of lipid domains. To achieve this goal, hybrid lipids were compared with domain-forming lipids, and unlike previous simulations, multiple nAChR oligomers were included as opposed to using only one nAChR molecule. Within coarse-grained (CG) model membranes, nAChRs consistently partitioned into flexible, liquid-disordered domains; boundary lipids were rich in DHA regardless of the number of nAChR molecules, but preventing domain formation also reduced the likelihood of DHA aggregating around nAChR. Taken together, our findings suggest that by inducing domain formation in membranes, DHA plays a critical role in the early stages of nAChR oligomerization.

At the neuromuscular junction (NMJ), the nicotinic acetylcholine receptor (nAChR) self-associates to give rise to rapid muscle movement. While lipid domains have maintained nAChR aggregates in vitro, their specific roles in nAChR clustering are currently unknown. In the present study, we carried out coarse-grained molecular dynamics simulations (CG-MD) of 1–4 nAChR molecules in two membrane environments: one mixture containing domain-forming, homoacidic lipids, and a second mixture consisting of heteroacidic lipids. Spontaneous dimerization of nAChRs was up to ten times more likely in domain-forming membranes; however, the effect was not significant in four-protein systems, suggesting that lipid domains are less critical to nAChR oligomerization when protein concentration is higher. With regard to lipid preferences, nAChRs consistently partitioned into liquid-disordered domains occupied by the omega-3 ( $$\omega$$ -3) fatty acid, docosahexaenoic acid (DHA); enrichment of DHA boundary lipids increased with protein concentration, particularly in homoacidic membranes. This result suggests dimer formation blocks access of saturated chains and cholesterol, but not polyunsaturated chains, to boundary lipid sites.

Reconstituted nicotinic acetylcholine receptors (nAChRs) exhibit significant gain-of-function upon addition of cholesterol to reconstitution mixtures, and cholesterol affects the organization of nAChRs within domain-forming membranes, but whether nAChR partitions to cholesterol-rich liquid-ordered ("raft" or lo) domains or cholesterol-poor liquid-disordered (ldo) domains is unknown. We use coarse-grained molecular dynamics simulations to observe spontaneous interactions of cholesterol, saturated lipids, and polyunsaturated (PUFA) lipids with nAChRs. In binary Dipalmitoylphosphatidylcholine:Cholesterol (DPPC:CHOL) mixtures, both CHOL and DPPC acyl chains were observed spontaneously entering deep "non-annular" cavities in the nAChR TMD, particularly at the subunit interface and the β subunit center, facilitated by the low amino acid density in the cryo-EM structure of nAChR in a native membrane. Cholesterol was highly enriched in the annulus around the TMD, but this effect extended over (at most) 5-10 Å. In domain-forming ternary mixtures containing PUFAs, the presence of a single receptor did not significantly affect the likelihood of domain formation. nAChR partitioned to any cholesterol-poor ldo domain that was present, regardless of whether the ldo or lo domain lipids had PC or PE headgroups. Enrichment of PUFAs among boundary lipids was positively correlated with their propensity for demixing from cholesterol-rich phases. Long n-3 chains (tested here with Docosahexaenoic Acid, DHA) were highly enriched in annular and non-annular embedded sites, partially displacing cholesterol and completely displacing DPPC, and occupying sites even deeper within the bundle. Shorter n-6 chains were far less effective at displacing cholesterol from non-annular sites.

Nicotinic acetylcholine receptors (nAChR) are pentameric ligand gated ion channels, critical to signaling across synapses and the neuro-muscular junction. While sensitive to boundary lipids, nAChR have been shown to be functionally dependent on cholesterol. This dependence on cholesterol has led to the hypothesis that nAChR resides within the cholesterol rich liquid ordered domains. Using the MARTINI force field, coarse-grained molecular dynamic simulations were preformed, with nAChRs in quasi-native ternary membranes. Native nAChR membrane composition has an abundance of polyunsaturated fatty acids (PUFAs), saturated fatty acids, and cholesterol. The two PUFAs chosen for these simulations were Docosahexaenoic acid and Linoleic acid. These simulations display nAChR consistently residing in the PUFA enriched disordered domain, remaining nearby the liquid ordered domain. Analysis of boundary lipid composition confirms nAChR boundary lipids are enriched in PUFAs. Further analysis of nAChR subunit-domain interaction show alpha subunits preference for cholesterol rich domains, while beta subunits show preference for PUFAs. Lastly, analysis shows PUFAs and cholesterol binding non-annularly nAChR. This study is being expanded to compare complex quasi-native synaptic and oocyte membranes. The oocyte membrane, in particular, is an optimal model for studying lipid-protein interactions, because it has a lower abundance of n-3 PUFAs compared to the neuron. From our simulations, we find that differences in membrane composition are especially noticeable around nAChRs. Given that nAChRs no longer exhibit partitioning preferences in oocyte membranes, our initial simulations suggest that oocytes do not provide a sufficiently native-like environment for nAChR.

The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric ligand gated ion channel found throughout the nervous system. At the neuromuscular junction, nAChRs cluster at a high density in order to transmit a fast and robust signal to the muscle. While nAChR clusters are stabilized by the dimerization of the intracellular protein, rapsyn, this dimerization process is extremely sensitive to changes in lipid environment. Neuronal membranes are rich in polyunsaturated lipids (PUFAs), cholesterol, and PE headgroups, with an overall composition similar to that of the Torpedo Californica electric organ. One PUFA in particular, Docosahexaenoic acid (DHA), is prevalent in both neuronal and Torpedo membranes, and is associated with domain formation in membranes. Through our preliminary simulations, we observed single nAChRs partitioning into the liquid-disordered (ld) domains, while remaining close to liquid-ordered (cholesterol-rich) domains. While researchers have speculated that nAChR clustering is facilitated by the formation of lipid domains, experiments investigating such lipid-protein interactions have been inconclusive. In the present study, we use coarse-grained molecular dynamics simulations to investigate the partitioning and clustering behavior of multiple nAChRs in a range of membrane environments with various amounts of intrinsic domain formation. For quasi-native membranes, nAChRs consistently partition into liquid-disordered phases, exhibiting a strong affinity for the domain interface and even dimerizing along well-defined interfaces. Oligomerization is not observed in membranes lacking domains. Boundary lipids are rich in polyunsaturated acyl chains regardless of the number of nAChR molecules, but preventing domain formation also reduces the likelihood of these acyl chains aggregating around nAChR. Taken together, these results imply that nAChR organization, oligomerization, and local environment is highly sensitive to membrane organization, with long-tailed PUFAs playing a critical role in both phase separation and protein clustering in neuronal membranes.

Nicotinic acetylcholine receptors (nAChRs) are pentameric Ligand Gated Ion Channels critical to signaling across synapses and the neuromuscular junction. nAChR function is particularly sensitive to the surrounding lipids, with numerous experimental studies showing native function of reconstituted nAChRs only in membranes with cholesterol. It has been expected that cholesterol serves as a boundary lipid by binding to the nAChR in annular and possibly non-annular (embedded) sites; given the expectation of annular cholesterol it was further hypothesized that nAChR likely partitions into liquid-ordered (raft) phases of domain-separated membranes, but this has not been observed experimentally in simple domain-forming mixtures containing cholesterol. Furthermore, although n-3 polyunsaturated fatty acids (PUFAs) are abundant in both the native nAChR torpedo membrane and the neuron, the role of these acyl chains in nAChR has not been studied experimentally. In the present research, we use Coarse-grained Molecular Dynamics Simulations via MARTINI to investigate spontaneous partitioning of nAChRs in domain-forming lipid mixtures based on the native torpedo lipid environment. We observe that, contrary to expectations, nAChR partitions into the liquid-disordered phase rich in n-3 PUFAs and low in cholesterol. When nAChR is partitioned into a cholesterol-poor liquid-disordered phase, binding of annular cholesterol is not observed, but cholesterol is stable in some non-annular embedded sites at some cholesterol concentrations. Most such non-annular sites seem to have a higher affinity for n-3 PUFAs than for cholesterol, however. One origin of nAChR's preference for the liquid-disordered phase becomes clear upon examining the equilibrated systems: the more flexible liquid-disordered phase can accommodate the deformation induced by the cone-shaped nAChR.

Nicotinic acetylcholine receptors (nAChRs) are pentameric Ligand Gated Ion Channels that are critical to signaling across synapses and the neuromuscular junction; such signaling is facilitated by high densities of nAChRs in the post-synaptic membrane. Organization of nAChRs, including partitioning behavior in membranes containing distinct lipid domains, is poorly characterized. Numerous experimental studies have shown nAChR gain-of-function likely caused by direct interactions with cholesterol, but a significant role for lipid domains has been suggested by nAChR gain-of-function upon cholesterol depletion. Furthermore, the opportunity for direct interactions will likely have a complex dependence on the extent of domain formation in the membrane, which has not been previously addressed. In the present research, we use Molecular Dynamics Simulations with coarse-grained resolution via the MARTINI model to investigate concentrations of cholesterol and other lipids local to nAChRs embedded in complex model membranes with a range of head groups and degrees of unsaturation. Cholesterol and unsaturated lipids are observed binding in deep 'non-annular' sites in the nAChR bundle (based on the 2BG9 cryo-EM structure), consistent with our previous predictions. nAChR partitions, however, into cholesterol-poor phases, resulting in dynamic exchange between cholesterol and unsaturated phospholipids, as well as a non-monotonic dependence of the number of direct cholesterol interactions on the cholesterol concentration in the membrane bulk.