Mapping of the Lipid-Binding Regions of the Antifungal Protein NFAP2 by Exploiting Model Membranes,Journal of Chemical Information and Modeling

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Mapping of the Lipid-Binding Regions of the Antifungal Protein NFAP2 by Exploiting Model Membranes
Journal of Chemical Information and Modeling ( IF 5.6 ) Pub Date : 2024-08-16 , DOI: 10.1021/acs.jcim.4c00229
Olivér Pavela _{1,

2} , Tünde Juhász ₁ , Liliána Tóth ₃ , András Czajlik _{4,

5} , Gyula Batta ₄ , László Galgóczy _{3,

6} , Tamás Beke-Somfai ₁

Affiliation

Fungal infections with high mortality rates represent an increasing health risk. The Neosartorya (Aspergillus) fischeri antifungal protein 2 (NFAP2) is a small, cysteine-rich, cationic protein exhibiting potent anti-Candida 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-Candida agents and studies aiming at future medical use of peptide-based natural antifungal compounds.

中文翻译：

通过利用模型膜绘制抗真菌蛋白 NFAP2 的脂质结合区域

高死亡率的真菌感染代表着越来越大的健康风险。Neosartorya （Aspergillus） fischeri 抗真菌蛋白 2 （NFAP2）是一种富含半胱氨酸的小阳离子蛋白，具有强大的抗念珠菌活性。作为潜在机制，孔的形成已经得到证明;然而，缺乏关于其膜破坏作用的分子水平细节。在此，我们使用结合计算和实验方法解决了 NFAP2 的脂质结合问题，该方法具有不同表面电荷特性的简单脂质组成。模拟结果揭示了带负电荷的模型膜的结合偏好，其中选择性由蛋白质结合位点富集的阴离子脂质成分介导，但也由两性离子脂质种类辅助。观察到由各种锚定接触引发的几种潜在结合途径，这导致了一种主要结合模式和一些变体，其中 NFAP2 驻留在膜表面。蛋白质柔性 N 末端部分的 ¹⁰区 NCPNNCKHKKG²⁰ 显示出插入脂质双层的潜力，其中二硫键稳定的短基序 ¹¹CPNNC¹⁵ 可能起关键作用。此外，包括 N 末端（残基 1-8）的起始区域在促进初始膜接触中发挥作用。此外，模拟还揭示了 Lys24、Lys32、Lys34 和 Trp42 等残基的个体作用。综合数据表明，溶液构象在膜相互作用时没有明显扰动，并且蛋白质的折叠部分也有助于稳定结合状态。数据还强调，NFAP2 与脂质囊泡的结合受离子强度等环境因素的敏感影响。阴离子脂质驱动的静电相互作用被发现是关键的，这解释了在高盐条件下观察到的膜活性降低。实验数据支持脂质选择性结合机制，并指出盐依赖性效应，特别是在低离子强度下蛋白质辅助囊泡聚集。我们的研究结果有助于开发基于 NFAP2 的抗念珠菌药物和旨在未来基于肽的天然抗真菌化合物的医学用途的研究。

更新日期：2024-08-16

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