Nawaz Nida, Nawaz Shiza, Hussain Athar, Anayat Maryam, Wen Sai, Wang Fenghuan
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing 100048, China.
School of Light Industry Science and Engineering, Beijing Technology & Business University (BTBU), Beijing 100048, China.
Int J Mol Sci. 2025 Sep 3;26(17):8579. doi: 10.3390/ijms26178579.
Endolysins, phage-derived enzymes capable of lysing bacterial cell walls, hold significant promise as novel antimicrobials against resistant Gram-positive and Gram-negative pathogens. In this study, we undertook an integrative approach combining extensive in silico analyses and experimental validation to characterize the novel endolysin LysPALS22. Initially, sixteen endolysin sequences were selected based on documented lytic activity and enzymatic diversity, and subjected to multiple sequence alignment and phylogenetic analysis, which revealed highly conserved catalytic and binding domains, particularly localized to the N-terminal region, underscoring their functional importance. Building upon these sequence insights, we generated three-dimensional structural models using Swiss-Model, EBI-EMBL, and AlphaFold Colab, where comparative evaluation via Ramachandran plots and ERRAT scores identified the Swiss-Model prediction as the highest quality structure, featuring over 90% residues in favored conformations and superior atomic interaction profiles. Leveraging this validated model, molecular docking studies were conducted in PyRx with AutoDock Vina, performing blind docking of key peptidoglycan-derived ligands such as N-Acetylmuramic Acid-L-Alanine, which exhibited the strongest binding affinity (-7.3 kcal/mol), with stable hydrogen bonding to catalytic residues ASP46 and TYR61, indicating precise substrate recognition. Visualization of docking poses using Discovery Studio further confirmed critical hydrophobic and polar interactions stabilizing ligand binding. Subsequent molecular dynamics simulations validated the stability of the LysPALS22-NAM-LA complex, showing minimal structural fluctuations, persistent hydrogen bonding, and favorable interaction energies throughout the 100 ns trajectory. Parallel to computational analyses, LysPALS22 was heterologously expressed in () and (), where SDS-PAGE and bicinchoninic acid assays validated successful protein production; notably, the -expressed enzyme displayed an increased molecular weight (~45 kDa) consistent with glycosylation, and achieved higher volumetric yields (1.56 ± 0.31 mg/mL) compared to (1.31 ± 0.16 mg/mL), reflecting advantages of yeast expression for large-scale production. Collectively, these findings provide a robust structural and functional foundation for LysPALS22, highlighting its conserved enzymatic features, specific ligand interactions, and successful recombinant expression, thereby setting the stage for future in vivo antimicrobial efficacy studies and rational engineering efforts aimed at combating multidrug-resistant Gram-negative infections.
内溶素是一种能够裂解细菌细胞壁的噬菌体衍生酶,作为对抗耐药革兰氏阳性和革兰氏阴性病原体的新型抗菌剂具有巨大潜力。在本研究中,我们采用了一种综合方法,将广泛的计算机分析和实验验证相结合,以表征新型内溶素LysPALS22。最初,基于已记录的裂解活性和酶多样性选择了16个内溶素序列,并进行了多序列比对和系统发育分析,结果显示高度保守的催化和结合结构域,特别是位于N端区域,突出了它们的功能重要性。基于这些序列见解,我们使用Swiss - Model、EBI - EMBL和AlphaFold Colab生成了三维结构模型,通过拉氏图和ERRAT分数进行比较评估,确定Swiss - Model预测为质量最高的结构,超过90%的残基处于有利构象且具有优越的原子相互作用特征。利用这个经过验证的模型,在PyRx中使用AutoDock Vina进行了分子对接研究,对关键的肽聚糖衍生配体如N - 乙酰胞壁酸 - L - 丙氨酸进行了盲对接,其表现出最强的结合亲和力(-7.3 kcal/mol),与催化残基ASP46和TYR61形成稳定的氢键,表明对底物的精确识别。使用Discovery Studio对对接姿势进行可视化进一步证实了稳定配体结合的关键疏水和极性相互作用。随后的分子动力学模拟验证了LysPALS22 - NAM - LA复合物的稳定性,在整个100 ns轨迹中显示出最小的结构波动、持续的氢键和有利的相互作用能。与计算分析并行,LysPALS22在()和()中进行了异源表达,其中SDS - PAGE和二辛可宁酸测定法验证了蛋白质的成功生产;值得注意的是,在()中表达的酶显示分子量增加(约45 kDa)与糖基化一致,并且与()(1.31±0.16 mg/mL)相比实现了更高的体积产量(1.56±0.31 mg/mL),反映了酵母表达在大规模生产方面的优势。总体而言,这些发现为LysPALS22提供了坚实的结构和功能基础,突出了其保守的酶学特征、特定的配体相互作用和成功的重组表达,从而为未来针对多重耐药革兰氏阴性感染的体内抗菌疗效研究和合理工程努力奠定了基础。