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

Abstract

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 A Ê 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 A Ê from the L3L4 calcium site. The close spacing and the difference in calcium af®nity 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-X n -G-X 2 -H-X-SNG) that contains conserved residues involved in dimerisation and catalysis.

Surface Loop Motion in FepA

“…Although the crystal structures of many E. coli outer membrane proteins are known (e.g., OmpF [7], LamB [37], OmpA [31], FepA [5], FhuA [11,23], phospholipase A2 [41], OmpT [43], and TolC [18]), the nature and extent of conformational motion within these proteins in vivo is unknown. FepAK332 resides in an unsolved region of L4 (5); K483 resides in L7, and only the ␣ and ␤ carbons of its side chain were crystallographically described, presumably because of the flexibility of the loop (Fig.…”

Phospholipase A in Gram-negative bacteria and its role in pathogenesis

“…Comparison of OMPLA amino acid sequences in these bacteria also revealed that 30 amino acid residues are completely conserved, indicating an important physiological role of this enzyme in Gram-negative bacteria (Brok et al, 1998). However, in a following study which involved sequence alignment of complete OMPLA sequences in 16 different species of Gram-negative bacteria, Snijder et al (2001) indicated that only 20 amino acid residues are absolutely conserved and another 17 are similar in the sequences of E. coli, Bordetella pertussis, Campylobacter coli, C. jejuni, Enterobacter agglomerans, H. pylori 2669551, H. pylori J99, Salmonella typhi, S. typhimurium, K. pneumoniae, N. gonorrhoeae, N. meningitidis, Pasteurella multocida, Proteus vulgaris, Y. pestis and Y. pseudotuberculosis, therefore yielding a homology of only 13?5 % (Fig. 4).…”

“…Several of the conserved residues are close in sequence, and form a complete and highly specific consensus sequence motif (YTQ-X n -G-X 2 -H-X-SNG), which proves to be much more specific than a motif based on the active-site residues alone as indicated in Fig. 4 (Snijder et al, 2001). …”

“…Crystallographic studies by Snijder et al (2001) revealed that the L3L4 site was found only in the structure of monomeric OMPLA, whereas the interfacial site (catalytic site) was only found in the dimeric enzyme, with no calcium binding observed at the L3L4 site. This indicates that calcium binding occurs in the L3L4 site of OMPLA monomers.…”

“…However, the calcium ion is displaced from this site when the dimers are formed, which indicates that calcium plays an important role in the formation of the active dimer from two inactive monomers. Furthermore, Snijder et al (2001) suggested that the L3L4 site could serve as a sink for calcium ions, and that it might complex with the LPS matrix, thus maintaining the barrier function of the outer membrane. They also indicated that the key elements of this model (calcium binding and activation) are conserved in Gramnegative bacteria, suggesting that OMPLA architecture in this group is most likely the same, and that its activity is calcium-and dimerization-dependent.…”