基于分子动力学模拟提高嗜热磷酸三酯酶样内酯酶非特异性底物活力的理论研究

doi:10.7503/cjcu20180445

摘要/Abstract

摘要：

采用分子动力学模拟和拉伸分子动力学模拟方法, 结合分子力学-广义玻恩表面积(MM-GB/SA)方法进行自由能计算和结构交互指纹分析, 研究了模拟过程中非特异性底物(对氧磷/内酯)分别与嗜热磷酸三酯酶样内酯酶(SsoPox)野生型和突变体(W263F/W263T)结合的构象变化, 分析了Loop8中重要残基Trp263的突变提高SsoPox非特异性底物活力的原因, 发现其能够影响门控残基Phe229的构象变化, 导致活性口袋入口变宽(Phe229与Tyr99之间的距离变大), 使对氧磷和内酯更容易结合到蛋白质的活性位点上; Asp256和Arg223形成盐桥的几率高于野生型(WT)SsoPox, 在Arg223(位于Loop7)的协助下质子更加高效地从活性中心的Asp256(位于Loop8)传递到溶剂中去, 因而能够提高SsoPox水解底物的效率. 通过比较2个野生型复合物的结构稳定性和结合自由能差异, 发现在模拟过程中SsoPox与内酯的复合物体系更加稳定并且具有更低的结合自由能, 有利于SsoPox识别底物并使其埋在活性部位的疏水环境中, 促进氢氧化物亲核进攻底物的亲电中心. 因此, 底物与酶稳定的相互作用可能是SsoPox具有天然内酯酶活性的原因之一.

关键词: 嗜热类磷酸三酯酶样内酯酶, 分子动力学模拟, 拉伸分子动力学模拟, 分子力学-广义玻恩表面积, 结构交互指纹分析

Abstract:

The structural changes of phosphotriesterase-like lactonase induced by paraoxon and undecanoic-γ-lactone binding to WT SsoPox and W263F/W263T mutant were compared using molecular dynamics simulation and steered molecular dynamics simulation combined with molecular mechanics-generalized Born/surface area(MM-GB/SA) and structural interaction fingerprints(SIFt) methods. The reasons that the important residue Trp263(located in Loop 8) in SsoPox could determine the enzymatic promiscuity were listed as follow, (1) the displacements in two mutant complex could lead to a widening of the active site entrance, which allowed for tighter fitting of the substrate into the enzyme’s active site; (2) the salt bridge between Asp256 and Arg223 in the two mutant complexes occurred with a higher probability compared to two WT SsoPox complexes, which could offer a potential pathway for proton relay after catalysis. The results of comparing conformational stabilities and binding free energies between WT SsoPox and W263F/W263T mutant indicated that the complex of SsoPox and lactone was more stable and had lower binding free energy during the simulation, which was beneficial to the recognition of the substrate and nucleophilic attack of the hydroxide on the electrophilic center of the substrate. Therefore, the stable interaction of substrate with enzyme may be a reason for the natural lactonase activity of SsoPox.

Key words: Phosphotriesterase-like lactonase, Molecular dynamics simulation, Steered molecular dynamics simulation, Molecular mechanics-generalized Born/surface area(MM-GB/SA), Structural interaction fingerprint

中图分类号:

TrendMD:

朱镜璇, 于正飞, 刘野, 詹冬玲, 韩佳睿, 田晓翩, 韩葳葳. 基于分子动力学模拟提高嗜热磷酸三酯酶样内酯酶非特异性底物活力的理论研究. 高等学校化学学报, 2019, 40(1): 138.

ZHU Jingxuan,YU Zhengfei,LIU Ye,ZHAN Dongling,HAN Jiarui,TIAN Xiaopian,HAN Weiwei. Exploration of Increasing the Non-specificity Substrates Activity for the Phosphotriesterase-like Lactonase Using Molecular Dynamics Simulations^†. Chem. J. Chinese Universities, 2019, 40(1): 138.

图/表 11

Fig.1 Population analysis of LUMO orbital for paraoxon(A) and undecanoic-γ-lactone(B)

Fig.2 All ligands located in active pocket

Table 1 Structural interaction fingerprints results(residues number according to PDB ID: 2VC7)

Amino acid	Interaction
Amino acid	Any	Backbone	Sidechain	Polar	Hydrophobcic	H-Bond acceptor	H-Bond donor	Aromatic	Charged
His24	0.72	0	0.72	0.28	0.44	0	0	0	0
His22	0.26	0	0.26	0.11	0.15	0	0	0	0
Tyr97	0.85	0	0.85	0.45	0.40	0	0.13	0.72	0
His170	0.81	0	0.81	0.34	0.47	0	0	0	0
Arg223	0.88	0	0.88	0.71	0.16	0	0.24	0	0.14
Asp256	0.85	0.68	0.85	0.67	0.18	0	0	0	0
Trp278	0.84	0	0.84	0.08	0.75	0	0	0.79	0
Trp263	0.89	0	0.89	0	0.88	0	0	0.88	0
Leu226	0.68	0	0.68	0	0.68	0	0	0	0
Cys258	0.47	0	0.47	0.25	0.24	0	0	0	0
Ile261	0.53	0.05	0.53	0.05	0.51	0	0	0	0
Thr265	0.31	0.21	0.31	0.20	0.13	0	0	0	0
Ala266	0.41	0.31	0.41	0	0.40	0	0	0	0
Leu274	0.24	0.13	0.24	0	0.21	0	0	0	0
Ala275	0.22	0.12	0.22	0	0.20	0	0	0	0
Leu228	0.38	0	0.38	0	0.38	0	0	0	0
Phe229	0.48	0	0.48	0	0.48	0	0	0.47	0
Tyr99	0.31	0	0.31	0.06	0.25	0	0	0.27	0
Val27	0.49	0	0.49	0	0.49	0	0	0	0
Leu72	0.48	0	0.48	0	0.47	0	0	0	0

Fig.3 Conformational changes in WT, W263F and W263T SsoPox during 100 ns MD(A1, A2) RMSD; (B1, B2) RMSF; (C1, C2) SASA; (A1, B1, C1) paraoxon; (A2, B2, C2) lactone.

Fig.4 3D structure of residues Tyr99 and Phe229

Fig.5 Distance between the Cα of Tyr99 and Phe229 in WT-paraoxon and W263F-paraoxon(A), WT-lactone and W263T-lactone(B) during 100 ns MD

Fig.6 3D structure for residues Arg223 and Asp256 with donor and acceptor atoms

Fig.7 Distance between the donor and acceptor atoms in WT-paraoxon and W263F-paraoxon(A), WT-lactone and W263T-lactone(B) during 100 ns MD

Fig.8 Decomposing the free energy contributions to the binding free energy of protein-ligand in a per-residue basis

Table 2 Calculate the binding free energy of a protein-ligand complex using MM-GB/SA methods

System	WT-lactone	W263T-lactone	WT-paraoxon	W263F-paraoxon
ΔE_ele/(kJ·mol^-1)	-47.72	-90.50	-15.64	-93.63
ΔE_vdw/(kJ·mol^-1)	-126.76	-129.39	-72.68	-101.62
ΔG_GB/(kJ·mol^-1)	83.97	102.50	42.49	109.23
ΔG_SA/(kJ·mol^-1)	-17.73	-18.69	-10.79	-16.18
Δ $G polar a$ /(kJ·mol^-1)	36.26	12.00	26.85	15.64
Δ $G nonpolar b$ /(kJ·mol^-1)	-144.49	-148.08	-83.47	-117.81
Δ $H MM ? GB / SA c$ /(kJ·mol^-1)	-108.23	-136.08	-56.62	-102.17
-TΔS/(kJ·mol^-1)	80.80	72.52	77.58	92.13
Δ $G bind d$ /(kJ·mol^-1)	-27.43	-63.57	20.95	-10.04
$k cat$ / $K m e$ /(s^-1·L·mol^-1)	(2.36±0.38)×10³	(3.49±1.13)×10⁵	(5.19±0.95)×10²	(1.21±0.26)×10⁴

Table 2 Calculate the binding free energy of a protein-ligand complex using MM-GB/SA methods

System	WT-lactone	W263T-lactone	WT-paraoxon	W263F-paraoxon
ΔE_ele/(kJ·mol^-1)	-47.72	-90.50	-15.64	-93.63
ΔE_vdw/(kJ·mol^-1)	-126.76	-129.39	-72.68	-101.62
ΔG_GB/(kJ·mol^-1)	83.97	102.50	42.49	109.23
ΔG_SA/(kJ·mol^-1)	-17.73	-18.69	-10.79	-16.18
Δ $G polar a$ /(kJ·mol^-1)	36.26	12.00	26.85	15.64
Δ $G nonpolar b$ /(kJ·mol^-1)	-144.49	-148.08	-83.47	-117.81
Δ $H MM ? GB / SA c$ /(kJ·mol^-1)	-108.23	-136.08	-56.62	-102.17
-TΔS/(kJ·mol^-1)	80.80	72.52	77.58	92.13
Δ $G bind d$ /(kJ·mol^-1)	-27.43	-63.57	20.95	-10.04
$k cat$ / $K m e$ /(s^-1·L·mol^-1)	(2.36±0.38)×10³	(3.49±1.13)×10⁵	(5.19±0.95)×10²	(1.21±0.26)×10⁴

Fig.9 SMD simulation of typical force profiles of paraoxon(A) and lactone(B) pulled out of the binding pocket along the unbinding pathway, the distance between the Cz of Tyr99 and Phe229 in WT-paraoxon and W263F-paraoxon(C), WT-lactone and W263T-lactone(D) and conformational changes of WT-paraoxon(E), W263F-paraoxon(F), WT-lactone(G), W263T-lactone(H) during 10 ns SMD

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