Solution structure of .gamma.1-H and .gamma.1-P thionins from barley and wheat endosperm determined by proton NMR: a structural motif common to toxic arthropod proteins

Abstract

The complete assignment of the proton NMR spectra of the homologous gamma 1-hordothionin and gamma 1-purothionin (47 amino acids, 4 disulfide bridges) from barley and wheat, respectively, has been performed by two-dimensional sequence-specific methods. A total of 299 proton-proton distance constraints for gamma 1-H and 285 for gamma 1-P derived from NOESY spectra have been used to calculate the three-dimensional solution structures. Initial structures have been generated by distance geometry methods and further refined by dynamical simulated annealing calculations. Both proteins show identical secondary and tertiary structure with a well-defined triple-stranded antiparallel beta-sheet (residues 1-6, 31-34, and 39-47), an alpha-helix (residues 16-28), and the corresponding connecting loops. Three disulfide bridges are located in the hydrophobic core holding together the alpha-helix and the beta-sheet and forming a cysteine-stabilized alpha-helical (CSH) motif. Moreover, a clustering of positive charges is observed on the face of the beta-sheet opposite to the helix. The three-dimensional structures of the gamma-thionins differ remarkably from plant alpha- and beta-thionins and crambin. However, they show a higher structural analogy with scorpion toxins and insect defensins which also present the CSH motif.

Expression of functional Raphanus sativus antifungal protein in yeast

“…It is noteworthy that Rs-AFP2 belongs to a family of plant proteins that comprises the y-thionins from cereals [1]. y-Thionins have recently been shown to have a three-dimensional structure similar to that of insect defensins [14]. The striking requirements of the MF~I prodomain for expression of both types of structurally related proteins may be explained by assuming that the pro-domain facilitates correct folding of the mature domain.…”

Solution structure of the variant‐3 neurotoxin from Centruroides sculpturatus Ewing

“…proteins (see Bruix et al, 1993;Bontems et al, 1991a, b; various references sited in these papers). The principal feature of this motif is an a-helical segment with the sequence -C-X-X-X-C-that is coupled to a P-strand -C-X-C-through disulfide bridges (amB).…”

Primary Structure of ω‐Hordothionin, a Member of a Novel Family of Thionins from Barley Endosperm, and Its Inhibition of Protein Synthesis in Eukaryotic and Prokaryotic Cell‐Free Systems

“…In fact, w-hordothionin, unlike yhordothionin, has been shown to exist in aqueous solution as a mixture of two dil'fesent proline ci.s-tvuns, as revealed by the analysis of its two-dimensional NMR spectra 1331. Both cu-hordothionin and 11-hordothionin share common structural motifs including the cysteine-stabilized a-helix [14]. The three-dimensional structural similarity of this new w-hordothionin [33] with ythionins [14], as well as with some scorpion toxins and insect defensins [17], suggests a common mode of functional activity.…”

“…and -ys43) [14]. Finally, group IV is represented by FST [21], the 10-kDa peptide [31], the potato p322 polypeptide [32] and RS-AFPI [19].…”

“…thionin (y,-P) from barley and wheat has been established [14] using two-dimensional NMR spectroscopy. These structures, which differ considerably from those obtained for the related cysteine-rich polypeptides a,-purothionin [I51 and crambin [16], show a great analogy with scorpion toxins and insect defensins [17].…”