Clustering of transmembrane proteins underlies a multitude of fundamental biological processes at the plasma membrane (PM) such as receptor activation, lateral domain formation, and mechanotransduction. The self-association of the respective transmembrane domains (TMDs) has also been suggested to be responsible for the micron-scaled patterns seen for integral membrane proteins in the budding yeast PM. However, the underlying interplay between the local lipid composition and the TMD identity is still not mechanistically understood. In this work, we combined coarse-grained molecular dynamics simulations of simplified bilayer systems with high-resolution live-cell microscopy to analyze the distribution of a representative helical yeast TMD from the PM sensor Slg1 within different lipid environments. In our simulations, we specifically evaluated the effects of acyl chain saturation and anionic lipid head groups on the association of two TMDs. We found that weak lipid−protein interactions significantly affect the configuration of TMD dimers and the free energy of association. Increased amounts of unsaturated phospholipids (PLs) strongly reduced the helix−helix interaction, while the presence of anionic phosphatidylserine (PS) hardly affected the dimer formation. We could experimentally confirm this surprising lack of effect of PS using the network factor, a mesoscopic measure of PM pattern formation in yeast cells. Simulations also showed that the formation of TMD dimers in turn increased the order parameter of the surrounding lipids and induced long-range perturbations in lipid organization. In summary, our results shed new light on the mechanisms of lipid-mediated dimerization of TMDs in complex lipid mixtures.

Sterols have been ascribed a major role in the organization of biological membranes, in particular for the formation of liquid ordered domains in complex lipid mixtures. Here, we employed molecular dynamics simulations to compare the effects of cholesterol and ergosterol as the major sterol of mammalian and fungal cells, respectively, on binary mixtures with 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) as a proxy for saturated lipids. In agreement with previous work, we observe that the addition of sterol molecules modifies the order of DPPC both in the gel phase and in the liquid phase. When disentangling the overall tilt angle and the structure of the tail imposed by trans/gauche configurations of torsion angles in the tail, respectively, a more detailed picture of the impact of sterols can be formulated, revealing, for example, an approximate temperature-concentration superposition ranging from the liquid to the gel phase. Furthermore, a new quantitative measure to identify the presence of collective sterol effects is discussed. Moreover, when comparing both types of sterols, addition of cholesterol has a noticeably stronger impact on phospholipid properties than that of ergosterol. The observed differences can be attributed to higher planarity of the cholesterol ring system. This planarity combined with an inherent asymmetry in its molecular interactions leads to better alignment and hence stronger interaction with saturated acyl chains. Our results suggest that the high order demonstrated for ergosterol in fungal plasma membranes must therefore be generated via additional mechanisms.

Calcium release-activated calcium (CRAC) channels open upon depletion of Ca from the endoplasmic reticulum, and when open, they are permeable to a selective flux of calcium ions. The atomic structure of Orai, the pore domain of CRAC channels, from Drosophila melanogaster has revealed many details about conduction and selectivity in this family of ion channels. However, it is still unclear how residues on the third transmembrane helix can affect the conduction properties of the channel. Here, molecular dynamics and Brownian dynamics simulations were employed to analyze how a conserved glutamate residue on the third transmembrane helix (E262) contributes to selectivity. The comparison between the wild-type and mutated channels revealed a severe impact of the mutation on the hydration pattern of the pore domain and on the dynamics of residues K270, and Brownian dynamics simulations proved that the altered configuration of residues K270 in the mutated channel impairs selectivity to Ca over Na. The crevices of water molecules, revealed by molecular dynamics simulations, are perfectly located to contribute to the dynamics of the hydrophobic gate and the basic gate, suggesting a possible role in channel opening and in selectivity function.

The master kinase LKB1 is a key regulator of se veral cellular processes, including cell proliferation, cell polarity and cellular metabolism. It phosphorylates and activates several downstream kinases, including AMP-dependent kinase, AMPK. Activation of AMPK by low energy supply and phosphorylation of LKB1 results in an inhibition of mTOR, thus decreasing energy-consuming processes, in particular translation and, thus, cell growth. LKB1 itself is a constitutively active kinase, which is regulated by posttranslational modifications and direct binding to phospholipids of the plasma membrane. Here, we report that LKB1 binds to Phosphoinositide-dependent kinase (PDK1) by a conserved binding motif. Furthermore, a PDK1-consensus motif is located within the kinase domain of LKB1 and LKB1 gets phosphorylated by PDK1 in vitro. In Drosophila, knockin of phosphorylation-deficient LKB1 results in normal survival of the flies, but an increased activation of LKB1, whereas a phospho-mimetic LKB1 variant displays decreased AMPK activation. As a functional consequence, cell growth as well as organism size is decreased in phosphorylation-deficient LKB1. Molecular dynamics simulations of PDK1-mediated LKB1 phosphorylation revealed changes in the ATP binding pocket, suggesting a conformational change upon phosphorylation, which in turn can alter LKB1’s kinase activity. Thus, phosphorylation of LKB1 by PDK1 results in an inhibition of LKB1, decreased activation of AMPK and enhanced cell growth.

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Mutual interactions between the transmembrane domains of membrane proteins and lipids on the bilayer properties has gained major interest. Most simulation studies of membranes rely on the Martini force field, which has proven extremely helpful in providing molecular insights into realistic systems. Accordingly, an evaluation of the accuracy of Martini is crucial to be able to correctly interpret the reported data. In this study, we combine atomistic and coarse-grained Martini simulations to investigate the properties of transmembrane domains (TMDs) in a model yeast membrane. The results show that the TMD binding state (monomeric, dimeric with positive or negative crossing angle) and the membrane composition significantly influence the properties around the TMDs and change TMD-TMD and TMD-lipid affinities. Furthermore, ergosterol (ERG) exhibits strong affinity to TMD dimers. Importantly, the right-handed TMD dimer configuration is stabilized via TMD-TMD contacts by addition of asymmetric anionic PS. The CG simulations corroborate many of these findings, with two notable exceptions: a systematic overestimation of TMD-ERG interaction and lack of stabilization of the right-handed TMD dimers with the addition of PS. Atomistic simulation results suggest that a meaningful comparison of dimer formation and experimentally-determined network factor may require to additionally take into account the precise conformation and thermodynamic relevance of multimeric TMD clusters.

Pluripotent stem cells can yield different cell types depending on a sequence of differentiation signals as it activates/deactivates functions and keeps a memory of previous inputs. Herein, we achieve pluripotency in synthetic cells with three dormant apo-metalloenzymes such that they can differentiate towards different fates depending on the sequence of specific metal ion transport with ionophores. In the first differentiation step, the selective transport of extracellular metal ion cofactors into pluripotent giant unilamellar vesicles (GUVs) differentially activates enzymatic pathways that give rise to an increase of intracellular pH, production of hydrogen peroxide, or cell lysis. Formerly added ionophores suppress transport with subsequent ionophores due to in membrane interactions between ionophores. Consequently, the addition of a second ionophore leads to a dampened response in the multipotent GUV and a third ionophore in no further response, reminiscent of a terminally differentiated GUV. Taken together, the pluripotent GUV differentiates into five final fates depending on the sequence of three ionophores by virtue of adaptive metal ion transport.

Cholesterol and ergosterol are two dominant sterols in the membranes of eukaryotic and yeast cells, respectively. Although their chemical structure is very similar, their impact on the structure and dynamics of membranes differs. In this work, we have explored the effect of these two sterols on binary mixtures with 1,2-dipalnitoyl-sn-glycerol-3-phosphocholine (DPPC) lipid bilayer at various sterol concentration and temperatures, employing molecular dynamics simulations. The simulations revealed that cholesterol has a stronger impact on the ordering of the lipid chains and leads to more condensed membranes with respect to ergosterol. This difference likely arises from a more planar structure of the ring part as well as the better alignment of cholesterol among the DPPC chains with respect to ergosterol. The degree of the planarity of the ring system affects the orientation of the methyl groups on the rough side and distribute the lipid chains on the two sides of the sterols differently. Similar to the structural observations, cholesterol also has a stronger influence on the dynamics, and consistently, establishes stronger DPPC-sterol interactions when compared to ergosterol. Although our findings are consistent with some previous simulations as well as recent experiments, they are at odds with some other studies. Therefore, the presented results may shed new lights on the impact of sterols on the saturated lipids bilayers with implications for binary mixtures of lipids as well as lipid rafts.SignificanceCholesterol and ergosterol are crucial lipid molecules of eukaryotic and prokaryotic cells, respectively, with an important role for the characteristics of the membranes. Surprisingly, many experimental studies have reported opposing results concerning their relative impact. Our work aims to understand the molecular mechanism behind the influence of these sterols on the properties of saturated DPPC chains via a systematic computational approach at atomic resolution. The results show that cholesterol has a higher impact on the ordering, condensing and dynamics of the lipid chains and closely interact with them due to its more planar structure as compared to ergosterol. These effects can have implications in lipid rafts and the interaction of therapeutic drugs with membranes.