In silico identification of genetic mutations conferring resistance to acetohydroxyacid synthase inhibitors: A case study of Kochia scoparia

Mutations that confer herbicide resistance are a primary concern for herbicide-based chemical control of invasive plants and are often under-characterized structurally and functionally. As the outcome of selection pressure, resistance mutations usually result from repeated long-term applications of herbicides with the same mode of action and are discovered through extensive field trials. Here we used acetohydroxyacid synthase (AHAS) of Kochia scoparia (KsAHAS) as an example to demonstrate that, given the sequence of a target protein, the impact of genetic mutations on ligand binding could be evaluated and resistance mutations could be identified using a biophysics-based computational approach. Briefly, the 3D structures of wild-type (WT) and mutated KsAHAS-herbicide complexes were constructed by homology modeling, docking and molecular dynamics simulation. The resistance profile of two AHAS-inhibiting herbicides, tribenuron methyl and thifensulfuron methyl, was obtained by estimating their binding affinity with 29 KsAHAS (1 WT and 28 mutated) using 6 molecular mechanical (MM) and 18 hybrid quantum mechanical/molecular mechanical (QM/MM) methods in combination with three structure sampling strategies. By comparing predicted resistance with experimentally determined resistance in the 29 biotypes of K. scoparia field populations, we identified the best method (i.e., MM-PBSA with single structure) out of all tested methods for the herbicide-KsAHAS system, which exhibited the highest accuracy (up to 100%) in discerning mutations conferring resistance or susceptibility to the two AHAS inhibitors. Our results suggest that the in silico approach has the potential to be widely adopted for assessing mutation-endowed herbicide resistance on a case-by-case basis.赋予除草剂抗性的突变是入侵植物基于除草剂的化学防控工作中的核心关切，这类突变的结构与功能特征往往尚未得到充分解析。作为选择压力作用的结果，抗性突变通常源于同一作用机制除草剂的长期重复施用，并通过大规模田间试验得以发现。本研究以地肤（Kochia scoparia）的乙酰羟酸合酶（acetohydroxyacid synthase, AHAS，缩写为KsAHAS）为例，证明了：若已知靶标蛋白的序列，即可采用基于生物物理学的计算方法，评估遗传突变对配体结合的影响，并鉴定出抗性突变。简言之，本研究通过同源建模、分子对接与分子动力学模拟，构建了野生型（wild-type, WT）及突变型KsAHAS-除草剂复合物的三维结构。针对两种AHAS抑制剂类除草剂——苯磺隆（tribenuron methyl）与噻吩磺隆（thifensulfuron methyl），本研究通过6种分子力学（molecular mechanical, MM）方法、18种混合量子力学/分子力学（quantum mechanical/molecular mechanical, QM/MM）方法，并结合3种结构采样策略，估算了它们与29种KsAHAS变体（1种野生型、28种突变型）的结合亲和力，从而得到其抗性谱特征。通过将预测的抗性结果与29个地肤田间种群生物型的实验测定抗性结果进行对比，本研究在所有测试方法中，筛选出了适配除草剂-KsAHAS体系的最优方法——即基于单结构的MM-PBSA方法，该方法在区分赋予两类AHAS抑制剂抗性或敏感性的突变时，准确率最高可达100%。本研究结果表明，该in silico方法具备逐案推广应用的潜力，可用于评估由突变赋予的除草剂抗性。