1. Specific binding sites for an antifungal plant defensin from Dahlia (Dahlia merckii) on fungal cells are required for antifungal activity
K Thevissen, R W Osborn, D P Acland, W F Broekaert Mol Plant Microbe Interact. 2000 Jan;13(1):54-61. doi: 10.1094/MPMI.2000.13.1.54.
Dm-AMP1, an antifungal plant defensin from seeds of dahlia (Dahlia merckii), was radioactively labeled with t-butoxycarbonyl-[35S]-L-methionine N-hydroxy-succinimi-dylester. This procedure yielded a 35S-labeled peptide with unaltered antifungal activity. [35S]Dm-AMP1 was used to assess binding on living cells of the filamentous fungus Neurospora crassa and the unicellular fungus Saccharomyces cerevisiae. Binding of [35S]Dm-AMP1 to fungal cells was saturable and could be competed for by preincubation with excess, unlabeled Dm-AMP1 as well as with Ah-AMP1 and Ct-AMP1, two plant defensins that are highly homologous to Dm-AMP1. In contrast, binding could not be competed for by more distantly related plant defensins or structurally unrelated antimicrobial peptides. Binding of [35S]Dm-AMP1 to either N. crassa or S. cerevisiae cells was apparently irreversible. In addition, whole cells and microsomal membrane fractions from two independently obtained S. cerevisiae mutants selected for resistance to Dm-AMP1 exhibited severely reduced binding affinity for [35S]Dm-AMP1, compared with wild-type yeast. This finding suggests that binding of Dm-AMP1 to S. cerevisiae plasma membranes is required for antifungal activity of this protein.
2. The three-dimensional solution structure of Aesculus hippocastanum antimicrobial protein 1 determined by 1H nuclear magnetic resonance
F Fant, W F Vranken, F A Borremans Proteins. 1999 Nov 15;37(3):388-403. doi: 10.1002/(sici)1097-0134(19991115)37:33.3.co;2-6.
Aesculus hippocastanum antimicrobial protein 1 (Ah-AMP1) is a plant defensin isolated from horse chestnuts. The plant defensins have been divided in several subfamilies according to their amino acid sequence homology. Ah-AMP1, belonging to subfamily A2, inhibits growth of a broad range of fungi. So far, a three-dimensional structure has been determined only for members of subfamilies A3 and B2. In order to understand activity and specificity of these plant defensins, the structure of a protein belonging to subfamily A2 is needed. We report the three-dimensional solution structure of Ah-AMP1 as determined from two-dimensional 1H nuclear magnetic resonance data. The structure features all the characteristics of the "cysteine-stabilized alpha beta-motif." A comparison of the structure, the electrostatic potential surface and regions important for interaction with the fungal receptor, is made with Rs-AFP1 (plant defensin of subfamily A3). Thus, residues important for activity and specificity have been assigned.
