Phospholipase A2Mechanism Of Interfacial Activation,An Interdiscliplinary Approach

Phospholipase A2 (PLA2) is an enzyme that hydrolyzes the sn-2-ester bond of membrane phospholipids and liberates arachidonic acid, which is converted to eicosanoids that act as potent mediators of inflammation and allergy. As such this enzyme plays a crucial role in many homeostatic physiological and immunologic processes and disease progression. PLA2s undergo substantial increase in activity upon binding to cellular membranes. This effect of interfacial activation is well recognized, yet its structural and physical aspects are poorly understood. In this work, we have employed the interdisciplinary methods of molecular biology, biochemistry, biophysics, bioinformatics and computational biology, in order to elucidate the structure-function relationships mediating the interfacial activation of human group IIA and group IB PLA2 isoforms. We have evaluated the structural and functional consequences of two conservative, single residue substitutions, located at key membrane-binding and substrate-binding positions of hIIA PLA2. We have also evaluated a human group IB fragment (hIBΔN10), missing the first 10 N-terminal residues which make up the N-terminal alpha helix, as well as a chimeric enzyme substituting the N-terminal alpha helix of hIB PLA2 with that from hIIA PLA2 (hIIA/IB PLA2). We have compared the engineered proteins against both the hIIA and hIB PLA2 native enzymes and their N-terminal peptides, N10-hIB and N10-hIIA, respectively. We have developed and used a novel multidisciplinary approach in order to position the segmentally labeled hIB PLA2 and hIIA/IB chimeric PLA2s at the membrane surface. The results of this work provide significant insight into the understanding of the physical aspects of interfacial activation by determining the precise membrane binding modes of PLA2 isoforms and identifying certain amino acid residues and whole protein segments that play key roles in membrane binding, activation, and involved allosteric conformational effects in PLA2s