Energetics of outer membrane phospholipase A (OMPLA) dimerization.

Abstract

Outer membrane phospholipase A (OMPLA) is a widely conserved transmembrane enzyme found in Gram-negative bacteria, and it is implicated in the virulence of a number of pathogenic organisms. The regulation of the protein's phospholipase activity is not well understood despite the existence of a number of high resolution structures. Previous biochemical studies have demonstrated that dimerization of OMPLA is a prerequisite for its phospholipase activity, and it has been shown in vitro that this dimerization is dependent on calcium and substrate binding. Therefore, to fully understand the regulation of OMPLA, it is necessary to understand the stability of the protein dimer and the extent to which it is influenced by its effector molecules. We have used sedimentation equilibrium analytical ultracentrifugation to dissect the energetics of Escherichia coli OMPLA dimerization in detergent micelles. We find that calcium contributes relatively little stability to the dimer, while interactions with the substrate acyl chain are the predominant force in stabilizing the dimeric conformation of the enzyme. The resulting thermodynamic cycle suggests that interactions between effector molecules are additive. These energetic measurements not only provide insight into the activation of OMPLA, but they also represent the first quantitative investigation of the association energetics of a transmembrane beta-barrel. This thermodynamic study allows us to begin to address the differences between protein-protein interfaces in transmembrane proteins with a helical fold to those of a beta-barrel fold and to more fully understand the forces involved in membrane protein interactions.