Author:

Keywords:

av-PFI, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, plant defensin, gamma-thionin, cysteine-stabilized alpha beta motif, simulated annealing, protein structure, NUCLEAR-MAGNETIC-RESONANCE, ANDROCTONUS-AUSTRALIS HECTOR, 3-DIMENSIONAL SOLUTION STRUCTURE, RESOLUTION SOLUTION STRUCTURE, SPIN COUPLING-CONSTANTS, DISTANCE GEOMETRY, PLANT DEFENSINS, SECONDARY STRUCTURE, GAMMA-PUROTHIONINS, BARLEY ENDOSPERM, Amino Acid Sequence, Defensins, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Plant Proteins, Protein Conformation, Seeds, Sequence Alignment, Sequence Homology, Amino Acid, Structure-Activity Relationship, Vegetables, 0304 Medicinal and Biomolecular Chemistry, 0601 Biochemistry and Cell Biology, 0605 Microbiology, 3101 Biochemistry and cell biology, 3107 Microbiology

Abstract:

Raphanus sativus Antifungal Protein 1 (Rs-AFP1) is a 51 amino acid residue plant defensin isolated from radish (Raphanus sativus L.) seeds. The three-dimensional structure in aqueous solution has been determined from two-dimensional 1H NMR data recorded at 500 MHz using the DIANA/REDAC calculation protocols. Experimental constraints consisted of 787 interproton distances extracted from NOE cross-peaks, 89 torsional constraints from 106 vicinal interproton coupling constants and 32 stereospecific assignments of prochiral protons. Further refinement by simulated annealing resulted in a set of 20 structures having pairwise root-mean-square differences of 1.35(+/- 0.35) A over the backbone heavy atoms and 2.11(+/- 0.46) A over all heavy atoms. The molecule adopts a compact globular fold comprising an alpha-helix from Asn18 till Leu28 and a triple-stranded beta-sheet (beta 1 = Lys2-Arg6, beta 2 = His33-Tyr38 and beta 3 = His43-Pro50). The central strand of this beta-sheet is connected by two disulfide bridges (Cys21-Cys45 and Cys25-Cys47) to the alpha-helix. The connection between beta-strand 2 and 3 is formed by a type VIa beta-turn. Even the loop (Pro7 to Asn17) between beta-strand 1 and the alpha-helix is relatively well defined. The structure of Raphanus sativus Antifungal Protein 1 features all the characteristics of the "cysteine stabilized alpha beta motif". A comparison of the complete structure and of the regions important for interaction with the fungal receptor according to a mutational study, is made with the structure of gamma-thionin, a plant defensin that has no antifungal activity. It is concluded that this interaction is both electrostatic and specific, and some possible scenarios for the mode of action are given.