Fungi are a newly emerging source of peptide antibiotics with therapeutic potential. Here, we report 17 new fungal defensin-like peptide (fDLP) genes and the detailed characterization of a corresponding synthetic fDLP (micasin) from a dermatophyte in terms of its structure, activity and therapeutic potential. NMR analysis showed that synthetic micasin adopts a “hallmark” cysteine-stablized α-helical and β-sheet fold. It was active on both Gram-positive and Gram-negtive bacteria, and importantly it killed two clinical isolates of methicillin-resistant Staphylococcus aureus and the opportunistic pathogen Pseudomonas aeruginosa at low micromolar concentrations. Micasin killed approximately 100% of treated bacteria within 3 h through a membrane nondisruptive mechanism of action, and showed extremely low hemolysis and high serum stability. Consistent with these functional properties, micasin increases survival in mice infected by the pathogenic bacteria in a peritonitis model. Our work represents a valuable approach to explore novel peptide antibiotics from a large resource of fungal genomes.

Scorpion K⁺ channel toxins and insect defensins share a conserved three-dimensional structure and related biological activities (defense against competitors or invasive microbes by disrupting their membrane functions), which provides an ideal system to study how functional evolution occurs in a conserved structural scaffold. Using an experimental approach, we show that the deletion of a small loop of a parasitoid venom defensin possessing the “scorpion toxin signature” (STS) can remove steric hindrance of peptide-channel interactions and result in a neurotoxin selectively inhibiting K⁺ channels with high affinities. This insect defensin-derived toxin adopts a hallmark scorpion toxin fold with a common cysteine-stabilized α-helical and β-sheet motif, as determined by nuclear magnetic resonance analysis. Mutations of two key residues located in STS completely diminish or significantly decrease the affinity of the toxin on the channels, demonstrating that this toxin binds to K⁺ channels in the same manner as scorpion toxins. Taken together, these results provide new structural and functional evidence supporting the predictability of toxin evolution. The experimental strategy is the first employed to establish an evolutionary relationship of two distantly related protein families.

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K+ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca2+ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 310-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K+ channel but is not capable of activating skeletal-type Ca2+ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K+ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon–intron structure, 3D-fold and function suggest that scorpion venom ICK-type K+ channel inhibitors and Ca2+ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K+ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.