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.