Effect of Lipid Asymmetry on the Enzymatic Activity of OmpLA

Compartmentalization of biochemical processes is one of the key concepts of life. Nature has realized with the advent of cellular membranes, which are composites of proteins, lipids and carbohydrates. Biological membranes support various physiological processes, including active transport, catalysis and cellular communication. In order to fulfill these manifold functions membranes display distinctly organized and chemically well-controlled structures. On the molecular level these include the formation of distinct lipid/protein domains and an asymmetric transmembrane distribution lipid species. In fact cells spend significant amount of energy to actively maintain this imbalance of lipids. Lipid asymmetry implies several questions of physiological relevance. Here we are interested in the coupling to protein function such as the outer membrane phospholipase A (OmpLA). OmpLA is an integral membrane enzyme located in the outer membrane of Gram-negative bacteria, such as Escherichia coli. Activation leads to a hydrolysis of phospholipids and is coupled to membrane perturbation that cause the formation of OmpLA dimers. Previous biophysical studies demonstrated that OmpLA readily forms dimers, when reconstituted into artificial bilayers with symmetric lipid composition. The enzymes natural environment, however, comprises of an asymmetric bilayer of lipopolysaccharide in the outer leaflet and phosphatidylethanolamine/phosphatidylglycerol in the inner leaflet. We therefore hypothesize that the asymmetric lipid environment serves to maintain the proteins monomeric dormant state. Membrane perturbation leads to a loss of lipid asymmetric which triggers the enzyme to clear the membrane from lipids flipped to the outer leaflet. We will reconstitute OmpLA in different symmetric and asymmetric artificial membranes and study its aggregation state and activity as a function of membrane structure. This will be achieved by a coupling of X-ray and neutron scattering techniques with fluorescent energy transfer measurements of labelled proteins. Scattering techniques will allow us to interrogate effects of membrane structure on protein dimerization. Fluorescence assays in turn will give us insight into aggregate form of OmpLA in the diverse lipid membranes. Research will be performed on chemically well-defined systems, i.e. unlike natural membranes, the lipid and protein composition will be well-known. This entails stringent experimental control and ensures full tractability of methods. Supported by an international network of researches with highly complementary expertise we will derive the significance of lipid asymmetry in OmpLA activation as a role model for the coupling of specific membrane composition to protein function. We expect that the project will form the basis for several follow-up research efforts in the field. Moreover, the designed artificial membranes might serve as prototypical platform for screening of membrane- active antimicrobial drugs.

All plasma membranes, i.e., the outer envelopes of cells, possess a high degree of asymmetry. This concerns the orientation of membrane proteins, for example, for the directed transport of molecules across the membrane, as well as the distribution of membrane lipids, i.e., the composition of the outer and inner membrane leaflets. The research project aimed to provide fundamental evidence for a coupling of this membrane asymmetry to the function of integral membrane proteins. To this end, the outer membrane phospholipase A (OmpLA), a structurally and functionally well-characterized integral enzyme, was selected. The task of this protein is the breakdown of phospholipids (hydrolysis) when they translocate to the 'wrong' side of the membrane. The goal of the project was to determine whether this process can be influenced by a change in membrane asymmetry. For this purpose, OmpLA was reconstituted into lipid vesicles with controlled symmetric or asymmetric lipid composition. Functional measurements showed that OmpLA responds to the differential lateral pressure fields in asymmetric membranes. Two main results are noteworthy: (i) The activity of the protein is slowed down by an increasing asymmetric composition of the leaflets in charge-neutral membranes. This can be explained by a simple membrane-mediated allosteric model, assuming that OmpLA must perform work against the external lateral membrane pressure to break down the phospholipids. (ii) In negatively charged membranes, an increased overall activity of the protein was observed. Interestingly, this can be at least partially understood by coupling of the ions to the stored spontaneous curvature of the lipids. This latter result provides a completely new understanding of the function of the plasma membrane and its coupling to the electrolyte composition of its aqueous environment. The generic nature of our results suggests that similar membrane-mediated mechanisms are likely to be effective for other membrane proteins. However, understanding the coupling between membrane protein function and membrane asymmetry is still in its infancy. The insights gained in this work can be considered as one of many steps that need to be taken towards a better understanding of membrane function. The fact that the function of membrane proteins is strongly influenced by their membrane environment and that asymmetric membranes behave very differently from symmetric ones, however, entails an immensely high potential for future studies with additional biochemical. Furthermore, most medical drugs target membrane proteins, providing another link to pharmaceutical sciences. It is expected that by expanding such efforts, a paradigm shift will occur in lipid-protein interactions and the function of the plasma membrane.