计算机模拟三氯生对FabI活性口袋及Loop区的调控机制

应用分子动力学模拟、蛋白质二级结构定义(DSSP)和口袋体积测量(POVME)计算方法分别研究了FabI(烯脂酰-ACP还原酶)-NAD+(氧化型烟酰胺腺嘌呤二核苷酸)二元复合物和FabI-NAD+-TCL(三氯生)三元复合物体系中活性口袋loop区构象、loop区二级结构、活性口袋体积以及底物(酰基不饱和链)通道随模拟时间的变化规律.研究表明,在FabI-NAD+-TCL三元复合物中,三氯生限制了活性口袋及底物通道的变化,活性口袋loop区呈现为规则、闭合的稳定构象,位于活性口袋正前方,常伴有螺旋二级结构的形成,从而使活性口袋体积变化较小、分布比较集中,底物通道较窄或处于关闭状态.而在FabI-NAD+二元复合物中,活性口袋loop区以无规则、开启的柔性构象存在,活性口袋体积变化较大,分布比较分散,底物通道明显较宽且不稳定.可见,三氯生能诱导FabI活性口袋及loop区的构象变化,使活性口袋构成了紧密的统一体、封闭了底物通道,从而阻碍了酰基不饱和链通过底物通道进入蛋白酶的催化中心,中断了该酶催化的还原反应和细菌脂肪酸合成循环.上述发现对深入认识三氯生的抗菌作用机制及相关药物的改良与设计具有重要的指导意义.

Simulated Mechanism of Triclosan in Modulating the Active Site and Loop of FabI by Computer

The impact of conformation of the active site loop, secondary structure, active site volume, and substrate (unsaturated acyl chain) channel as a function of simulation time caused by the FabI (enoyl-ACP reductase) inhibitor of triclosan were studied by molecular dynamics simulations, define secondary structure of proteins (DSSP), and pocket volume measurer (POVME). Triclosan restricted the changes of the active site and substrate channel of the FabI-NAD⁺-TCL (NAD⁺: nicotinamide adenine dinucleotide, TCL: triclosan) ternary complex. The active site loop formed an ordered, closed, and stable conformation, and was commonly associated with a helical structure in front of the active site. This made the active site volume change little, the volume distribution concentrated and the substrate channel size narrowed and almost closed. However, the active site loop was disordered, open, and flexible in the FabI-NAD⁺ binary complex. The changes of active site volume and distribution in the binary system were larger and more disperse than those in the ternary system. The substrate channel size in the binary system widened and became unstable. Triclosan induced residues of the active site and active site loop of FabI and made the ternary system more closed, which blocked the unsaturated acyl chains from getting into the catalytic center of FabI through the substrate channel, interrupted the reduction reaction and the elongation cycle of fatty acid synthesis. These results aid our understanding of potent inhibitory activity of triclosan and related compounds.