基于TEMβ-内酰胺酶突变与变构关系分析的抗生素耐药机制

Antibiotic resistance mechanism of TEM β-lactamase: Mutations induced and conformational fluctuations of it's structure

目的基于一系列计算生物信息学方法,探索TEMβ-内酰胺酶突变导致的构象变构与抗生素耐药性的关系。方法首先,采用直接耦合分析方法,预测TEM结构中决定其构象的直接和间接的强耦合残基对;其次,采用分子动力学模拟和蛋白质构象阻挫分析方法分析耐药性突变残基参与的耦合残基对在蛋白质构象变化中的作用;最后,采用蛋白质成药性结合位点预测方法Fd-DCA,预测TEM表面潜在的成药性变构位点。结果通过共耦合分析方法发现238-环结构域与Ω-环结构域内部或结构域之间,以及TEM-52的突变残基E104K、G238S和M182T之间存在最强的耦合关系,分子动力学证实这些耦合关系在蛋白质结构与功能维持中具有极为重要的作用。最后,在TEM表面发现了一个成药性变构位点,该位点与Ω-环结构域也强烈的耦合,说明可以通过针对该位点设计药物,间接控制TEM的构象结构,从而减小其对抗生素的识别能力,为抵御TEM引起的耐药研究带来新的研究思路。结论耐药性残基突变会通过打破原有的耦合关系,从而大幅度的影响蛋白质构象稳定性,增加TEM结构的柔性,帮助TEM更加容易识别和水解抗生素药物,产生耐药性。

Objective Based on a series of computational bioinformatics and molecular modeling methods,antibiotic resistance mechanism of TEM β-lactamase was explored by studying mutations induced conformational fluctuations of its structure.Methods Firstly,direct coupling analysis was used to predict the direct and indirect coupled residue pairs which determined the conformations of TEM.Then molecular dynamic simulation and conformational frustration estimation were employed to analyze the conformational fluctuations induced by the resistance mutations involved in coupled residue pairs.Finally,druggable biding site identification method,named as Fd-DCA,was adopted to search the TEM surface to find the potential anti-resistance drug design binding sites.Results The strongest couplings were identified to be located within and between the domains of 238-1oop and Ω-loop,as well as among the locations of mutant residues of TEM-52,i.e.,E104K,G238S and M182T.The molecular conformational change simulation and frustration estimation proved that these couplings played important roles in the formation of the conformations of TEM.A novel druggable allosteric binding site,which was strongly coupled with Ω-loop,was identified eventually.This opened the new way for designing anti-resistance drugs against β-lactamase.Conclusion The resistance mutations of TEM could extremely disturb the stability of these residues involved in couplings and increased the structural flexibility of TEM,leading to obvious conformational change which could make TEM easier to recognize and hydrolyze antibiotics.