Structural investigations of calcium binding and its role in activity and activation of outer membrane phospholipase A from Escherichia coli

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that catalyses the hydrolysis of phospholipids. Enzymatic activity is regulated by reversible dimerisation and calcium-binding. We have investigated the ro le of calcium by X-ray crystallography. In monomeric OMPLA, one calcium ion binds between two external loops (L3L4 site) at 10 Angstrom from the activ e site. After dimerisation, a new calcium-binding site (catalytic site) is formed at the dimer interface in the active site of each molecule at 6 Angs trom from the L3L4 calcium site. The close spacing and the difference in ca lcium affinity of both sites suggests that the L3L4 site may function as a storage site for a calcium ion, which relocates to the catalytic site upon dimerisation. A. sequence alignment demonstrates conservation of the cataly tic calcium site but evolutionary variation of the L3L4 site. The residues in the dimer interface are conserved as well, suggesting that all outer mem brane phospholipases require dimerisation and calcium in the catalytic site for activity. For this family of phospholipases, we have characterised a c onsensus sequence motif (YTQ-X-11-G-X-2 -H-X-SNG) that contains conserved r esidues involved in dimerisation and catalysis. (C) 2001 Academic Press.