Solution structure of porcine pancreatic phospholipase A₂ complexed with micelles and a competitive inhibitor

Summary

The three-dimensional structure of porcine pancreatic PLA₂ (PLA₂), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 ϕ torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1–62 and 72–124 is 0.75±0.09 Å for the backbone atoms and 1.14±0.10 Å for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA₂ and PLA₂ in the ternary complex is 3.5 Å for the backbone atoms and 4.0 Å for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal α-helix. Whereas free in solution Ala¹, Leu² and Trp³ are disordered, with the α-amino group of Ala¹ pointing out into the solvent, in the ternary complex these residues have an α-helical conformation with the α-amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA₂. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA₂ in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA₂.