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

• Published in: Journal of molecular biology 2001-06, Vol.309 (2), p.477-489
• Main Authors: Snijder, H.J, Kingma, R.L, Kalk, K.H, Dekker, N, Egmond, M.R, Dijkstra, B.W
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - metabolism; Binding Sites; Calcium - metabolism; Calcium - pharmacology; calcium-binding; Consensus Sequence; Conserved Sequence; crystal structure; Crystallography, X-Ray; dimerisation; Dimerization; Enzyme Activation - drug effects; Escherichia coli; Escherichia coli - enzymology; Evolution, Molecular; membrane enzyme; Models, Molecular; Molecular Sequence Data; OMPLA protein; phospholipase; Phospholipases A - chemistry; Phospholipases A - metabolism; Phospholipases A1; Protein Binding; Protein Structure, Quaternary; Sequence Alignment
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2001.4675

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 role of calcium by X-ray crystallography. In monomeric OMPLA, one calcium ion binds between two external loops (L3L4 site) at 10 Å from the active 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 Å from the L3L4 calcium site. The close spacing and the difference in calcium 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 catalytic calcium site but evolutionary variation of the L3L4 site. The residues in the dimer interface are conserved as well, suggesting that all outer membrane phospholipases require dimerisation and calcium in the catalytic site for activity. For this family of phospholipases, we have characterised a consensus sequence motif (YTQ-Xn-G-X2-H-X-SNG) that contains conserved residues involved in dimerisation and catalysis.