Elucidating the Membrane Binding Process of a Disordered Protein: Dynamic Interplay of Anionic Lipids and the Polybasic Region.

Intrinsically disordered regions of proteins are responsible for many biological processes such as in the case of liver kinase B1 (LKB1)-a serine/threonine kinase relevant for cell proliferation and cell polarity. LKB1 becomes fully activated upon recruitment to the plasma membrane by binding of its disordered C-terminal polybasic motif consisting of eight lysines/arginines to phospholipids. Here, we present extensive molecular dynamics (MD) simulations of the polybasic motif interacting with a model membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl phosphatidic acid (PA) and cell culture experiments. Protein-membrane binding effects are due to the electrostatic interactions between the polybasic amino acids and PAs. For significant binding, the first three lysines turn out to be dispensable, which was also recapitulated in cell culture using transfected GFP-LKB1 variants. LKB1-membrane binding results in nonmonotonous changes in the structure of the protein as well as the membrane, in particular, accumulation of PAs and reduced thickness at the protein-membrane contact area. The protein-lipid binding turns out to be highly dynamic due to an interplay of PA-PA repulsion and protein-PA attraction. The thermodynamics of this interplay is captured by a statistical fluctuation model, which allows the estimation of both energies. Quantification of the significance of each polar amino acid in the polybasic provides detailed insights into the molecular mechanism of protein-membrane binding of LKB1. These results can likely be transferred to other proteins, which interact by intrinsically disordered polybasic regions with anionic membranes.

Rapid and reversible optical switching of cell membrane area by an amphiphilic azobenzene.

Cellular membrane area is a key parameter for any living cell that is tightly regulated to avoid membrane damage. Changes in area-to-volume ratio are known to be critical for cell shape, but are mostly investigated by changing the cell volume via osmotic shocks. In turn, many important questions relating to cellular shape, membrane tension homeostasis and local membrane area cannot be easily addressed because experimental tools for controlled modulation of cell membrane area are lacking. Here we show that photoswitching an amphiphilic azobenzene can trigger its intercalation into the plasma membrane of various mammalian cells ranging from erythrocytes to myoblasts and cancer cells. The photoisomerization leads to a rapid (250-500 ms) and highly reversible membrane area change (ca 2 % for erythrocytes) that triggers a dramatic shape modulation of living cells.

Phosphorylation of LKB1 by PDK1 Inhibits Cell Proliferation and Organ Growth by Decreased Activation of AMPK.

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.

The impact of bilayer composition on the dimerization properties of the Slg1 stress sensor TMD from a multiscale analysis.

The impact of mutual interactions between the transmembrane domains of membrane proteins and lipids on bilayer properties has gained major attraction. 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 the Martini force field 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 and 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 a strong affinity to TMD dimers. Importantly, the right-handed TMD dimer configuration is stabilized via TMD-TMD contacts by the addition of asymmetric anionic phosphatidylserine (PS). The coarse-grained 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.

Lipid-Mediated Association of the Slg1 Transmembrane Domains in Yeast Plasma Membranes.

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.

Effect of Cholesterol Versus Ergosterol on DPPC Bilayer Properties: Insights from Atomistic Simulations.

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-3-phosphocholine (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.

Molecular Dynamics Simulations of Orai Reveal How the Third Transmembrane Segment Contributes to Hydration and Ca2+ Selectivity in Calcium Release-Activated Calcium Channels.

Calcium release-activated calcium (CRAC) channels open upon depletion of Ca2+ 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 Ca2+ 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.