Membrane curvature allosterically regulates the phosphatidylinositol cycle, controlling its rate and acyl-chain composition of its lipid intermediates.

Signaling 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 ϵ (DGKϵ) and liposomes, we studied the activity and acyl-chain specificity of an enzyme of the phosphatidylinositol (PI) cycle, DGKϵ. By systematically varying the model membrane lipid composition and physical properties, we found that DGKϵ 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 DGKϵ activity and acyl-chain specificity. Moreover, in a fine-tuned interplay between the enzyme and the membrane, DGKϵ favored the formation of structures with greater Gaussian curvature. DGKϵ does not bear a regulatory domain, and these findings reveal the importance of membrane curvature in regulating DGKϵ 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.

Thermodynamics of Methyl-ß-cyclodextrin-Induced Lipid Vesicle Solubilization: Effect of Lipid Headgroup and Backbone.

The low aqueous solubility of phospholipids makes necessary the use of lipid carriers in studies ranging from lipid traffic and metabolism to the engineering of model membranes bearing lipid transverse asymmetry. One particular lipid carrier that has proven to be particularly useful is methyl-ß-cyclodextrin (MßCD). To assess the interaction of MßCD with structurally different phospholipids, the present work reports the results of isothermal titration calorimetry in conjunction with dynamic light scattering measurements. The results showed that the interaction of MßCD with large unilamellar vesicles composed of a single type of lipid led to the solubilization of the lipid vesicle and, consequently, the complexation of MßCD with the lipids. This interaction is dependent on the nature of the lipid headgroup, with a preferable interaction with phosphatidylglycerol in comparison to phosphatidylcholine. It was also possible to show a role played by the phospholipid backbone in this interaction. In many cases, the differences in the transfer energy between one lipid and another in going from a bilayer to a cyclodextrin-bound state can be qualitatively explained by the energy required to extract the lipid from a bilayer. In all cases, the data showed that the solubilization of the vesicles is entropically driven with a large negative ΔCp, suggesting a mechanism dependent on the hydrophobic effect.