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

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.钙离子（Calcium ions, Ca²+）在产黄青霉（Penicillium chrysogenum）富含半胱氨酸的阳离子抗真菌蛋白PAF的毒性机制中发挥关键作用：胞外高浓度钙离子会以浓度依赖的方式，降低其对敏感模式真菌粗糙脉孢霉（Neurospora crassa）的毒性。然而，目前关于钙离子的影响机制细节，以及PAF在真菌细胞外的钙离子结合能力（这可能是其活性丧失的诱因）的研究仍较为匮乏。本研究借助核磁共振（nuclear magnetic resonance, NMR）、等温滴定量热法及分子动力学（molecular dynamics, MD）模拟，证实PAF可弱特异性结合钙离子。通过PAF的分子动力学模拟，我们预测其C端存在一个主要的钙离子结合位点，涉及天冬氨酸53（Asp53）与天冬氨酸55（Asp55），而天冬氨酸19（Asp19）被认为是潜在的钙离子结合位点。我们先前的研究显示，将Asp19突变为丝氨酸后，PAF的钙离子结合能力几乎不受影响，但却导致其抗真菌活性完全丧失。本次研究中，我们替换了PAF C端的天冬氨酸残基，并构建了丝氨酸双突变体PAF^D53S/D55S。与野生型PAF相比，PAF^D53S/D55S的特异性钙离子结合亲和力显著下降，但其抗真菌活性得以保留。为深入解析钙离子相互作用的细节，我们对游离态及结合钙离子的PAF与PAF^D53S/D55S开展了核磁共振及分子动力学结构与动力学研究。尽管我们发现这些蛋白质变体及其钙离子复合物之间存在一定差异，但这些效应无法解释此前观察到的钙离子对PAF活性的调控作用。综上，PAF可在C端选择性结合钙离子；然而，该钙离子结合似乎并未直接参与此前报道的PAF抗真菌活性调控过程。