Edited by Paul E. FraserSignaling events at membranes are often mediated by membrane lipid composition or membrane physical properties. These membrane properties could act either by favoring the membrane binding of downstream effectors or by modulating their activity. Several proteins can sense/generate membrane physical curvature (i.e. shape). However, the modulation of the activity of enzymes by a membrane's shape has not yet been reported. Here, using a cell-free assay with purified diacylglycerol kinase ⑀ (DGK⑀) and liposomes, we studied the activity and acyl-chain specificity of an enzyme of the phosphatidylinositol (PI) cycle, DGK⑀. By systematically varying the model membrane lipid composition and physical properties, we found that DGK⑀ has low activity and lacks acyl-chain specificity in locally flat membranes, regardless of the lipid composition. On the other hand, these enzyme properties were greatly enhanced in membrane structures with a negative Gaussian curvature. We also found that this is not a consequence of preferential binding of the enzyme to those structures, but rather is due to a curvature-mediated allosteric regulation of DGK⑀ activity and acylchain specificity. Moreover, in a fine-tuned interplay between the enzyme and the membrane, DGK⑀ favored the formation of structures with greater Gaussian curvature. DGK⑀ does not bear a regulatory domain, and these findings reveal the importance of membrane curvature in regulating DGK⑀ activity and acyl-chain specificity. Hence, this study highlights that a hierarchic coupling of membrane physical property and lipid composition synergistically regulates membrane signaling events. We propose that this regulatory mechanism of membrane-associated enzyme activity is likely more common than is currently appreciated.

Atomic resolution map of the soluble amyloid beta assembly (Aβn) “toxic surfaces” that facilitate the early pathogenic events in Alzheimer's disease (AD).

Caffeine is a small amphiphilic molecule, which is widely consumed as a stimulant to prevent fatigue, but is also used as a common drug adjuvant in modern medicine. Here, we show that caffeine interacts with unsaturated lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By combining X-ray diffraction and molecular dynamics simulations, we present evidence that caffeine partitions in lipid membranes and locates at the head group-tail group interface of the bilayers. By attracting water molecules from neighboring lipid molecules, it leads to the formation of "water pockets", i.e., a local increase of water density at this interface. Through this mechanism, caffeine leads to an overall decrease of the gauche defect density in the membranes and an increase of membrane thickness, indicating a loss of membrane fluidity. These non-specific membrane interactions may increase the efficacy of analgesic drugs through changes in the bioavailability and rate of metabolism of these drugs.

With the advent of polymyxin B (PmB) resistance in bacteria, the mechanisms for mcr-1 resistance are of crucial importance in the design of novel therapeutics. The mcr-1 phenotype is known to decrease membrane charge and increase membrane packing by modification of the bacterial outer membrane. We used X-ray diffraction, Molecular Dynamics simulations, electrochemistry, and leakage assays to determine the location of PmB in different membranes and assess membrane damage. By varying membrane charge and lipid tail packing independently, we show that increasing membrane surface charge promotes penetration of PmB and membrane damage, whereas increasing lipid packing decreases penetration and damage. The penetration of the PmB molecules is well described by a phenomenological model that relates an attractive electrostatic and a repulsive force opposing insertion due to increased membrane packing. The model applies well to several gram-negative bacterial strains and may be used to predict resistance strength.

In the brain, α‐synuclein (aSN) partitions between free unbound cytosolic and membrane bound forms modulating both its physiological and pathological role and complicating its study due to structural heterogeneity. Here, we use an interdisciplinary, synergistic approach to characterize the properties of aSN:lipid mixtures, isolated aSN:lipid co‐structures, and aSN in mammalian cells. Enabled by the isolation of the membrane‐bound state, we show that within the previously described N‐terminal membrane anchor, membrane interaction relies both on an N‐terminal tail (NTT) head group layer insertion of 14 residues and a folded‐upon‐binding helix at the membrane surface. Both binding events must be present; if, for example, the NTT insertion is lost, the membrane affinity of aSN is severely compromised and formation of aSN:lipid co‐structures hampered. In mammalian cells, compromised cooperativity results in lowered membrane association. Thus, avidity within the N‐terminal anchor couples N‐terminal insertion and helical surface binding, which is crucial for aSN membrane interaction and cellular localization, and may affect membrane fusion.

In our recent Communications Biology article, we reported on the biophysical mechanism of resistance for polymyxin antibiotics in bacterial membranes. The emergence of plasmid-borne colistin resistance poses a threat to our last line of defense against many pathogens. Here, we outline the current understanding of mcr-1-mediated polymyxin resistance, and propose future directions for membrane-targeting antibiotic research. Polymyxin antibiotics, such as colistin, have important roles in both medicine and agriculture. However, the use of polymyxins for the latter endangered its use in the former. The first transmissible plasmid-bearing resistance to colistin was reported by Liu and colleagues in 2016, and isolates of Escherichia coli harboring this resistance have been found in livestock across southeastern Asia 1. Expression of the resistance gene, mcr-1, causes modification of lipid A in the bacterial outer surface, resulting in reduced affinity for polymyxins. Worryingly, the gene can be readily passed between different bacterial strains making widespread polymyxin resistance inevitable 2. Wang and colleagues reported on the global distribution of mcr-1 and showed the resistance gene is currently present across five continents, as shown in Fig. 1 (ref. 3). Polymyxin antibiotics kill bacteria by damaging their cell membranes, but now bacteria have figured out how to inhibit this process. Biophysical tools, such as diffraction and molecular dynamics computer simulations, have provided important insights into these mechanisms 4,5. In order to prolong the lifespan of current polymyxins and develop new ones, it is critical to gain a detailed understanding of the biophysics of polymyxin-bacteria interactions. How do polymyxins damage bacterial membranes? The earliest high-resolution studies showed that polymyxins can kill bacteria by puncturing holes into their outer surface, and causing leakage of internal contents 6-8. Polymyxin B and colistin are both cationic antimicrobial peptides that are attracted to the net anionic bacterial outer membrane, resulting in an electrostatic attraction between drug and target. The polymyxin creates local curvature in the membrane, while the membrane itself repels the insertion process. At a critical point, the hydrophobic tail of the polymyxin can insert into the bacterial membrane and create a lipid defect by separating lipids away from one another. When fully inserted into the

Cortisone is an injected anti-inflammatory drug that can cause painful side effects known as “steroid flares” which are caused by cortisone crystallizing at the injection site. We used molecular dynamics simulations and X-ray diffraction to study the interaction of cortisone with model lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at drug concentrations from 0 mol% to 50 mol%. Cortisone was found to partition in the lipid bilayer and locate in the hydrophilic to hydrophobic interface of the membranes. Cortisone strongly affects the integrity of the membrane, as quantified by a decreased membrane thickness, increased area per lipid, and decreased lipid tail order parameters. At cortisone concentrations of more than 20 mol%, signals from crystallized cortisone were observed. These crystallites are embedded in the bilayers and orient with the membranes. While the cortisone molecules align parallel to the bilayers at low concentrations, they start to penetrate the hydrophobic core at higher concentrations. Trans-membrane crystallites start to nucleate when the membrane thickness has decreased such that cortisone molecules in the different leaflets can find partners from the opposite leaflet resulting in a non-zero density of cortisone molecules in the bilayer center. We suggest that the lipid bilayer provides a site for cortisone crystallization.

Amyloid-β aggregates play a causative role in Alzheimer’s disease. These aggregates are a product of the physical environment provided by the basic neuronal membrane, composed of a lipid bilayer. The intrinsic properties of the lipid bilayer allow amyloid-β peptides to nucleate and form well-ordered cross-β sheets within the membrane. Here, we correlate the aggregation of the hydrophobic fragment of the amyloid-β protein, Aβ25-35, with the hydrophobicity, fluidity, and charge density of a lipid bilayer. We summarize recent biophysical studies of model membranes and relate these to the process of aggregation in physiological systems.