Calcium binding of the antifungal protein PAF: Structure, dynamics and function aspects by NMR and MD simulations

Calcium ions (Ca(2+)) play an important role in the toxicity of the cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum: high extracellular Ca(2+) 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 Ca(2+) ion impact and the Ca(2+) 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 Ca(2+) ions. MD simulations of PAF predicted one major Ca(2+) binding site at the C-terminus involving Asp53 and Asp55, while Asp19 was considered as putative Ca(2+) binding site. The exchange of Asp19 to serine had little impact on the Ca(2+) 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 PAF(D53S/D55S). The specific Ca(2+) binding affinity of PAF(D53S/D55S) decreased significantly if compared to PAF, whereas the antifungal activity was retained. To understand more details of Ca(2+) interactions, we investigated the NMR and MD structure/dynamics of the free and Ca(2+)-bound PAF and PAF(D53S/D55S). Though we found some differences between these protein variants and the Ca(2+) complexes, these effects cannot explain the observed Ca(2+) influence. In conclusion, PAF binds Ca(2+) ions selectively at the C-terminus; however, this Ca(2+) binding does not seem to play a direct role in the previously documented modulation of the antifungal activity of PAF.