Interactions of phosphoinositide specific phospholipase C with a lipid layer for structural and functional studies

Phosphoinositide-specific phospholipase C (PLC) is an intensively studied family of enzymes constituting a junction between trans-membrane signal transduction processes and phosphoinositide lipid signalling. PLCs are activated in response to stimulation of cell surface receptors at the plasma membrane, and the signals are carried downstream by other transducers. PLCs catalyse the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol and inositol 1,4,5-trisphosphate, which are both well known intracellular second messengers. This study mainly focused on the PLCε sub-family which is closely linked to the Ras oncogene and may play a role in tumorigenesis and development. The functions and regulatory mechanism of PLCε are not yet understood in detail. To address these issues activity and structural studies were performed. Activity studies were carried out in vivo using cell lines and in vitro using lipid vesicles in a model system. The latter was designed to study protein-protein and lipid-protein interactions using PLC purified to homogeneity and guanosine triphosphatases (GTPases) prenylated in vitro. Evidence was found for a direct interaction between the GTPases and the PLC that mediated activation of the phospholipase. Furthermore, the correlation between PLC activity and substrate presentation in lipid vesicles of various sizes and lipid compositions was analysed. For the first time, PLC activity was found to depend upon the electrostatic potential and the stored elastic curvature stress of the lipid bilayer of the vesicles. The binding and the activation process between GTPase (using H-Ras) and PLCε was also investigated at a molecular level in vitro. Functional studies were carried out using Förster resonance energy transfer (FRET) to determine if PLCε undergoes a conformational change upon H-Ras binding. This would distinguish whether conformational change or translocation of PLCε to the membrane interface (where GTPases are localised) is most likely to be the key event during PLCε activation; no conformational change was observed. Electron crystallographic structural studies, in which two-dimensional protein crystals are grown on a lipid monolayer followed by electron microscopy, were attempted. The aim was to retrieve structural information in a functional state that resembles the natural one. Protein and lipid monolayer compositions (lipid proportions, lipid concentration, protein concentration and incubation time) were screened to identify conditions where specific protein-lipid interaction would favour twodimensional crystal formation. The protein was expressed with a His6-tag that allowed specific binding to nickel chelating lipids included in the lipid monolayer. In addition, catalytically inactive PLCε mutants were generated and their ability to bind PtdIns(4,5)P2, and thereby to drive the crystallisation process, was investigated. Conditions that led to protein-lipid binding, but not to two-dimensional crystallisation, were identified.