Mapping of the Lipid-Binding Regions of the Antifungal Protein NFAP2 by Exploiting Model Membranes

Fungal infections with high mortality rates represent an increasing health risk. The <i>Neosartorya (Aspergillus) fischeri</i> antifungal protein 2 (NFAP2) is a small, cysteine-rich, cationic protein exhibiting potent anti-<i>Candida</i> activity. As the underlying mechanism, pore formation has been demonstrated; however, molecular level details on its membrane disruption action are lacking. Herein, we addressed the lipid binding of NFAP2 using a combined computational and experimental approach to simple lipid compositions with various surface charge properties. Simulation results revealed binding preferences for negatively charged model membranes, where selectivity is mediated by anionic lipid components enriched at the protein binding site but also assisted by zwitterionic lipid species. Several potential binding routes initiated by various anchoring contacts were observed, which resulted in one main binding mode and a few variants, with NFAP2 residing on the membrane surface. Region ¹⁰NCPNNCKHKKG²⁰ of the flexible N-terminal part of the protein showed potency to insert into the lipid bilayer, where the disulfide bond-stabilized short motif ¹¹CPNNC¹⁵ could play a key role. In addition, several areas, including the beginning of the N-terminal (residues 1-8), played roles in facilitating initial membrane contacts. Besides, individual roles of residues such as Lys24, Lys32, Lys34, and Trp42 were also revealed by the simulations. Combined data demonstrated that the solution conformation was not perturbed markedly upon membrane interaction, and the folded part of the protein also contributed to stabilizing the bound state. Data also highlighted that the binding of NFAP2 to lipid vesicles is sensitively affected by environmental factors such as ionic strength. Electrostatic interactions driven by anionic lipids were found pivotal, explaining the reduced membrane activity observed under high salt conditions. Experimental data supported the lipid-selective binding mechanisms and pointed to salt-dependent effects, particularly to protein-assisted vesicle aggregation at low ionic strength. Our findings can contribute to the development of NFAP2-based anti-<i>Candida</i> agents and studies aiming at future medical use of peptide-based natural antifungal compounds.