Engineering dynamic gates in binding pocket of penicillin G acylase to selectively degrade bacterial signaling molecules

(01-mutants_design.tar.gz) Mutants design: Input structures of ecPGA from the PDB database (PDB IDs: 1GK9, 1GM7, and 1GM9), processed to resemble wild-type state, repaired by RepairPDB module of FoldX 4 Double-point mutants preparation, analysis and filtering: text files including configuration for FoldX 4 inputs and outputs of CAVER 3.02 calculations on FoldX 4 PDB files of ecPGA double-point mutants input configuration file and TransportTools library 0.9.4 calculations outputs generated based on the inputs produced in step 01 and 02 above CSV files containing complete information about FoldX 4 stability prediction and geometrical properties from CAVER 3.02 and TransportTools library version 0.9.4 for ecPGA double-point mutants Triple-point mutant preparation, analysis and filtering: text files including configuration for FoldX 4 inputs and outputs of CAVER 3.02 calculations on FoldX 4 PDB files of ecPGA triple-point mutants input configuration file and TransportTools library 0.9.4 calculations outputs generated based on the inputs produced in step 01 and 02 above CSV files containing complete information about FoldX 4 stability prediction and geometrical properties from CAVER 3.02 and TransportTools library version 0.9.4 for ecPGA triple-point mutants (02-docking.tar.gz) Preparation of protein-ligand complexes using molecular docking for wild-type ecPGA and 6 best designed triple-point mutants with 6 various bacterial signaling molecules: PDB files of ligand, PDBQT files of the receptor and PDB files of the complexes selected from docking experiment: Full names of presented protein variants:ecPGA_wt, wild-type Escherichia coli penicillin G acylaseLAF, Phe138αLeu & Met142αAla & Ile177βPhe ecPGA variant internally referred as 1GK9_Repair_22LSF, Phe138αLeu & Met142αSer & Ile177βPhe ecPGA variant internally referred as 1GK9_Repair_98MAF, Phe138αMet & Met142αAla & Ile177βPhe ecPGA variant internally referred as 1GK9_Repair_23MSF, Phe138αMet & Met142αSer & Ile177βPhe ecPGA variant internally referred as 1GK9_Repair_99VAF, Phe138αVal & Met142αAla & Ile177βPhe ecPGA variant internally referred as 1GK9_Repair_30YAF, Phe138αTyr & Met142αAla & Ile177βPhe ecPGA variant internally referred as 1GK9_Repair_33Full names of presented AHLs:C06, N-hexanoyl-L-homoserine lactone;C06-3O, N-3-oxo-hexanoyl-L-homoserine lactone;C08, N-octanoyl-L-homoserine lactone;C08-3O, N-3-oxo-octanoyl-L-homoserine lactone;C10, N-decanoyl-L-homoserine lactone;C12-3O, N-3-oxo-dodecanoyl-L-homoserine lactone (03-protein_ligand_MDs.tar.gz) Ligand-enzyme complexes molecular dynamics for wild-type ecPGA and 6 best designed triple-point mutants with 6 various bacterial signaling molecules: Force field parameters in Amber format Input coordinates *.inpcrd, parameters *.parm7 and *.pdb files for each complex ready for simulation in Amber Amber input files *.in for minimization, equilibration and production runs Restart files for each stage of the minimization, equilibration and production runs in Amber *.rst format Simulation output files for each stage of the minimization, equilibration and production runs in Amber *.mdout format Output files generated during post-processing of production runs trajectories in a form of text files generated by cpptraj (04-free_enzymes_MDs.tar.gz) Free enzymes molecular dynamics of 3 best triple-point ecPGA (VAF, YAF and MSF) mutants prioritized based on protein-ligand molecular dynamics simulations and experimental assays: Force field parameters and input coordinates *.inpcrd, parameters *.parm7 and *.pdb files for each complex ready for simulation in Amber format Amber input files *.in for minimization, equilibration and production runs Restart files for each stage of the minimization, equilibration and production runs in Amber *.rst format Simulation output files for each stage of the minimization, equilibration and production runs in Amber *.mdout format Post-processing analysis of generated trajectories: Text files with distances, CSV files containing result of PCA and clustering, PNG files with clustered PCA results Inputs and outputs of MDpocket analysis and visualization of the pocket frequency grid as an isomesh CAVER input configuration files in text format, CAVER output data including parsed CSV and text files for visualization of entrance opening time evolution and cavity profiles inspection cpptraj generated text files including RMSD, distances and chi1 angles measurements All plots were generated using matplotlib or seaborn Python libraries. Figures containing structural representations were generated using PyMOL 2.0.1. (05-ecPGA_VAF_YAF_MSF_penG_MDs.tar.gz) PenG-enzyme complexes molecular dynamics for wild-type ecPGA and 3 best designed triple-point mutants (VAF, YAF, MSF): PenG force field parameters in Amber (GAFF) format Input coordinates *.inpcrd, parameters *.parm7 and *.pdb files for each complex ready for simulation in Amber Amber input files *.in for minimization, equilibration and production runs Restart files for each stage of the minimization, equilibration and production runs in Amber *.rst format Simulation output files for each stage of the minimization, equilibration and production runs in Amber *.mdout format and analysis output files generated during post-processing of production runs trajectories in a form of text files Reactive Stabilization Score [RSS] statistics summarized in CSV files(01-mutants_design.tar.gz) 突变体设计： 从蛋白质数据库（Protein Data Bank，PDB）获取的大肠杆菌青霉素G酰基转移酶（ecPGA）输入结构（PDB编号：1GK9、1GM7及1GM9），经处理以模拟野生型状态，并通过FoldX 4的RepairPDB模块完成修复。 双点突变体制备、分析与筛选： 包含FoldX 4配置文件的文本文件； ecPGA双点突变体经FoldX 4处理后的PDB文件上运行CAVER 3.02的计算输入与输出文件； 基于上述01、02步骤生成的输入文件，得到的输入配置文件以及TransportTools库0.9.4的计算输出结果； 包含ecPGA双点突变体的FoldX 4稳定性预测信息，以及CAVER 3.02与TransportTools库0.9.4版本所得几何特性完整信息的CSV文件。 三点突变体制备、分析与筛选： 包含FoldX 4配置文件的文本文件； ecPGA三点突变体经FoldX 4处理后的PDB文件上运行CAVER 3.02的计算输入与输出文件； 基于上述01、02步骤生成的输入文件，得到的输入配置文件以及TransportTools库0.9.4的计算输出结果； 包含ecPGA三点突变体的FoldX 4稳定性预测信息，以及CAVER 3.02与TransportTools库0.9.4版本所得几何特性完整信息的CSV文件。 (02-docking.tar.gz) 分子对接制备野生型ecPGA与6株最优设计三点突变体及6种细菌信号分子的蛋白-配体复合物： 配体的PDB文件、受体的PDBQT文件，以及从对接实验中筛选得到的复合物PDB文件： 所展示的蛋白变体完整名称：ecPGA_wt，野生型大肠杆菌青霉素G酰基转移酶（ecPGA）；Phe138αLeu、Met142αAla及Ile177βPhe ecPGA变体，内部命名为1GK9_Repair_22LSF；Phe138αLeu、Met142αSer及Ile177βPhe ecPGA变体，内部命名为1GK9_Repair_98MAF；Phe138αMet、Met142αAla及Ile177βPhe ecPGA变体，内部命名为1GK9_Repair_23MSF；Phe138αMet、Met142αSer及Ile177βPhe ecPGA变体，内部命名为1GK9_Repair_99VAF；Phe138αVal、Met142αAla及Ile177βPhe ecPGA变体，内部命名为1GK9_Repair_30YAF；Phe138αTyr、Met142αAla及Ile177βPhe ecPGA变体，内部命名为1GK9_Repair_33。 所展示的酰基高丝氨酸内酯（AHLs）完整名称：C06：N-己酰基-L-高丝氨酸内酯；C06-3O：N-3-氧代-己酰基-L-高丝氨酸内酯；C08：N-辛酰基-L-高丝氨酸内酯；C08-3O：N-3-氧代-辛酰基-L-高丝氨酸内酯；C10：N-癸酰基-L-高丝氨酸内酯；C12-3O：N-3-氧代-十二烷酰基-L-高丝氨酸内酯。 (03-protein_ligand_MDs.tar.gz) 野生型ecPGA与6株最优设计三点突变体结合6种细菌信号分子的配体-酶复合物分子动力学模拟： Amber格式的力场参数文件； 每个复合物用于Amber模拟的输入坐标文件*.inpcrd、参数文件*.parm7及PDB文件； 用于最小化、平衡及生产模拟的Amber输入文件*.in； Amber格式*.rst的最小化、平衡及生产模拟各阶段的重启文件； Amber格式*.mdout的最小化、平衡及生产模拟各阶段的模拟输出文件； 通过cpptraj生成的文本文件形式的生产模拟轨迹后处理输出文件。 (04-free_enzymes_MDs.tar.gz) 基于蛋白-配体分子动力学模拟与实验测定筛选出的3株最优ecPGA三点突变体（VAF、YAF及MSF）的游离酶分子动力学模拟： Amber格式的力场参数文件以及每个复合物用于Amber模拟的输入坐标文件*.inpcrd、参数文件*.parm7及PDB文件； 用于最小化、平衡及生产模拟的Amber输入文件*.in； Amber格式*.rst的最小化、平衡及生产模拟各阶段的重启文件； Amber格式*.mdout的最小化、平衡及生产模拟各阶段的模拟输出文件； 轨迹后处理分析： 包含距离数据的文本文件、包含主成分分析（PCA）与聚类结果的CSV文件、展示聚类后PCA结果的PNG文件； MDpocket分析的输入输出文件，以及以等值网格形式展示的口袋频率网格可视化文件； CAVER输入配置文本文件、CAVER输出数据（包括用于可视化入口开放时间演化与空腔轮廓检查的已解析CSV及文本文件）； 通过cpptraj生成的包含RMSD、距离及chi1角测量结果的文本文件。 所有图表均通过matplotlib或seaborn Python库生成。包含结构可视化的图稿通过PyMOL 2.0.1生成。 (05-ecPGA_VAF_YAF_MSF_penG_MDs.tar.gz) 野生型ecPGA与3株最优设计三点突变体（VAF、YAF、MSF）结合青霉素G（penicillin G，PenG）的配体-酶复合物分子动力学模拟： Amber（GAFF）格式的PenG力场参数文件； 每个复合物用于Amber模拟的输入坐标文件*.inpcrd、参数文件*.parm7及PDB文件； 用于最小化、平衡及生产模拟的Amber输入文件*.in； Amber格式*.rst的最小化、平衡及生产模拟各阶段的重启文件； Amber格式*.mdout的最小化、平衡及生产模拟各阶段的模拟输出文件，以及以文本文件形式生成的生产模拟轨迹后处理输出文件； 汇总于CSV文件中的反应稳定评分（Reactive Stabilization Score，RSS）统计数据。