SOLUTION STRUCTURE OF PORCINE PANCREATIC PHOSPHOLIPASE A(2) COMPLEXED WITH MICELLES AND A COMPETITIVE INHIBITOR

The three-dimensional structure of porcine pancreatic PLA(2) (PLA(2)), 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 phi 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 Angstrom for the backbone atoms and 1.14 +/- 0.10 Angstrom for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA(2) and PLA(2) in the ternary complex is 3.5 Angstrom for the backbone atoms and 4.0 Angstrom 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 alpha-helix. Whereas free in solution Ala(1), Leu(2) and Trp(3) are disordered, with the alpha-amino group of Ala(1) pointing out into the solvent, in the ternary complex these residues have an alpha-helical conformation with the alpha-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 PLA(2). Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA(2) 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 st role in the interfacial activation of PLA(2).