Date Published: October 15, 2018
Publisher: Public Library of Science
Author(s): Ádám Fizil, Christoph Sonderegger, András Czajlik, Attila Fekete, István Komáromi, Dorottya Hajdu, Florentine Marx, Gyula Batta, Eugene A. Permyakov.
Calcium ions (Ca2+) play an important role in the toxicity of the cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum: high extracellular Ca2+ levels reduce the toxicity of PAF in the sensitive model fungus Neurospora crassa in a concentration dependent way. However, little is known about the mechanistic details of the Ca2+ ion impact and the Ca2+ binding capabilities of PAF outside the fungal cell, which might be the reason for the activity loss. Using nuclear magnetic resonance (NMR), isothermal titration calorimetry and molecular dynamics (MD) simulations we demonstrated that PAF weakly, but specifically binds Ca2+ ions. MD simulations of PAF predicted one major Ca2+ binding site at the C-terminus involving Asp53 and Asp55, while Asp19 was considered as putative Ca2+ binding site. The exchange of Asp19 to serine had little impact on the Ca2+ binding, however caused the loss of antifungal activity, as was shown in our recent study. Now we replaced the C-terminal aspartates and expressed the serine variant PAFD53S/D55S. The specific Ca2+ binding affinity of PAFD53S/D55S decreased significantly if compared to PAF, whereas the antifungal activity was retained. To understand more details of Ca2+ interactions, we investigated the NMR and MD structure/dynamics of the free and Ca2+-bound PAF and PAFD53S/D55S. Though we found some differences between these protein variants and the Ca2+ complexes, these effects cannot explain the observed Ca2+ influence. In conclusion, PAF binds Ca2+ ions selectively at the C-terminus; however, this Ca2+ binding does not seem to play a direct role in the previously documented modulation of the antifungal activity of PAF.
The small antifungal protein PAF is secreted by the filamentous ascomycete Penicillium chrysogenum. Since PAF shows no toxic effect on mammalian cells it represents a promising bio-molecule to prevent the growth of human and plant pathogenic fungi[2, 3]. The structures of PAF and related proteins generally consist of five antiparallel β-strands and the β-barrel tertiary structures are stabilized by three or four disulphide bonds that results in extreme temperature, pH and even protease stability[1, 4, 5]. Therefore, they may be excellent examples for rational drug design to develop new antifungal molecules. In order to understand the relationship between their biological activity and structural properties we have examined several of these proteins recently, especially PAF from the β-lactam antibiotics producer Penicillium chrysogenum. The solution structure of PAF was determined and its interesting dynamical behaviour was described previously[5, 6]. In the course of our intensive studies addressing the mechanistic function of this protein, we showed that PAF acts in a very complex way including activities at the outer fungal cell layers and also inside the target cell[1, 7, 8]. The perturbation of intracellular calcium (Ca2+) homeostasis was found to be closely associated with its toxic function[9, 10]. Ca2+ plays an essential role in all organisms in intracellular signalling and acts as an important second messenger in numerous signal transduction pathways that regulate various cellular responses. In the PAF-sensitive model fungi Neurospora crassa and Aspergillus niger, PAF elicits an immediate and sustained elevation of the cytosolic free Ca2+ [Ca2+]c concentration that is triggered by an influx of extracellular Ca2+ ions. In consequence, the PAF treated fungal cells are disturbed in polar growth and cell proliferation, and show apoptotic markers[1, 7, 9]. There is increasing evidence that the perturbation of the fungal [Ca2+]c homeostasis is a common mechanism that is also responsible for the growth inhibitory activity of other antifungal proteins, e.g. the antifungal protein AFPNN5353 of Aspergillus giganteus, several plant defensins and the antifungal hexapeptide RKKWFW (PAF26).
In this study, we investigated the hypothesis that binding of extracellular Ca2+ to PAF might be the reason for the loss of its antifungal activity. Our investigations shed new light on the Ca2+ binding ability outside the fungal cell and its impact on the structure-function relation of the antifungal protein PAF, which may support our understanding of the mode of action of other PAF- related antifungal proteins from filamentous fungi.