Penicillium antifungal protein (PAF) is a promising antimycotic without toxic effects on mammalian cells and therefore may represent a drug candidate against the often lethal Aspergillus infections that occur in humans. The pathogenesis of PAF on sensitive fungi involves G‐protein coupled signalling followed by apoptosis. In the present study, the solution structure of this small, cationic, antifungal protein from Penicillium chrysogenum is determined by NMR. We demonstrate that PAF belongs to the structural classification of proteins fold class of its closest homologue antifungal protein from Aspergillus giganteus. PAF comprises five β‐strands forming two orthogonally packed β‐sheets that share a common interface. The ambiguity in the assignment of two disulfide bonds out of three was investigated by NMR dynamics, together with restrained molecular dynamics calculations. The clue could not be resolved: the two ensembles with different disulfide patterns and the one with no S–S bond exhibit essentially the same fold. 15N relaxation dispersion and interference experiments did not reveal disulfide bond rearrangements via slow exchange. The measured order parameters and the 3.0 ns correlation time are appropriate for a compact monomeric protein of this size. Using site‐directed mutagenesis, we demonstrate that the highly‐conserved and positively‐charged lysine‐rich surface region enhances the toxicity of PAF. However, the binding capability of the oligosaccharide/oligonucleotide binding fold is reduced in PAF compared to antifungal protein as a result of less solvent‐exposed aromatic regions, thus explaining the absence of chitobiose binding. The present study lends further support to the understanding of the documented substantial differences between the mode of action of two highly homologous antifungal proteins.

BackgroundSmall, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses.ResultsThe APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays.ConclusionThis study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure–function analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0586-4) contains supplementary material, which is available to authorized users.

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Antimicrobial proteins (AMPs) are widely distributed in nature. In higher eukaryotes, AMPs provide the host with an important defence mechanism against invading pathogens. AMPs of lower eukaryotes and prokaryotes may support successful competition for nutrients with other microorganisms of the same ecological niche. AMPs show a vast variety in structure, function, antimicrobial spectrum and mechanism of action. Most interestingly, there is growing evidence that AMPs also fulfil important biological functions other than antimicrobial activity. The present review focuses on the mechanistic function of small, cationic, cysteine-rich AMPs of mammals, insects, plants and fungi with antifungal activity and specifically aims at summarizing current knowledge concerning additional biological properties which opens novel aspects for their future use in medicine, agriculture and biotechnology.