SOLUTION STRUCTURE OF PORCINE PANCREATIC PHOSPHOLIPASE A(2) COMPLEXEDWITH 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 i nhibitor, 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 c onstraints. A total of 18 structures was calculated using a combined a pproach of distance geometry and restrained molecular dynamics. The at omic 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 a veraged 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 Angstro m for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important d ifference is found for the first three residues of the N-terminal alph a-helix. Whereas free in solution Ala(1), Leu(2) and Trp(3) are disord ered, with the alpha-amino group of Ala(1) pointing out into the solve nt, in the ternary complex these residues have an alpha-helical confor mation with the alpha-amino group buried inside the protein. As a cons equence, the important conserved hydrogen bonding network which is als o seen in the crystal structures is present only in the ternary comple x, but not in free PLA(2). Thus, the NMR structure of the N-terminal r egion (as well as the calcium-binding loop and the surface loop) of PL A(2) in the ternary complex resembles that of the crystal structure. C omparison of the NMR structures of the free enzyme and the enzyme in t he ternary complex indicates that conformational changes play st role in the interfacial activation of PLA(2).