NMR SOLUTION STRUCTURE OF THE PATHOGENESIS-RELATED PROTEIN P14A

The nuclear magnetic resonance (NMR) structure of the 15 kDa pathogene sis-related protein P14a, which displays antifungicidal activity and i s induced in tomato leaves as a response to pathogen infection, was de termined using N-15/C-13 doubly labeled and unlabeled protein samples. In all, 2030 conformational constraints were collected as input for t he distance geometry program DIANA. After energy-minimization with the program OPAL the 20 best conformers had an average root-mean-square d eviation value relative to the mean coordinates of 0.88 Angstrom for t he backbone atoms N, C-alpha and C', and 1.30 Angstrom for all heavy a toms. P14a contains four alpha-helices (I to IV) comprising residues 4 to 17, 27 to 40, 64 to 72 and 93 to 98, a short 3(10)-helix of residu es 73 to 75 directly following helix III, and a mixed, four-stranded b eta-sheet with topology +3x, -2x, +1, containing the residues 24-25, 5 3 to 58, 104 to 111 and 117 to 124. These regular secondary structure elements form a novel, complex alpha+beta topology in which the alpha- helices I, III and IV and the 3(10)-helix are located above the plane defined by the beta-sheet, and the alpha-helix II lies below this plan e. The alpha-helices and beta-strands are thus arranged in three stack ed layers, which are stabilized by two distinct hydrophobic cores asso ciated with the two layer interfaces, giving rise to an ''alpha-beta-a lpha sandwich''. The three-dimensional structure of P14a provides init ial leads for identification of the so far unknown active sites' and t he mode of action of the protein, which is of direct interest for the generation of transgenic plants with improved host defense properties. (C) 1997 Academic Press Limited.