光谱法结合计算模拟探究胡桃醌与脯氨酰顺反异构酶1的相互作用

Interaction between Juglone and Peptidyl-prolyl Cis-trans Isomerase NIMA-interacting 1 by Spectroscopic Combined with Computational Simulation

胡桃醌为胡桃科核桃属植物胡桃楸(Jugland mandshurica Maxim)和核桃(Juglans regia L)中存在的重要活性物质,脯氨酰顺反异构酶1(Pin1)为细胞内主要起信号传导作用的小分子蛋白。为探究胡桃醌对Pin1的抑制机理,通过定点突变、计算模拟以及多种光谱学技术研究了胡桃醌与Pin1的相互作用。荧光光谱显示,胡桃醌可以有效地淬灭Pin1内源荧光。293 K温度时,其淬灭常数(Ksv)为1.36×10^(4) L/mol,结合常熟(Ka)为2.32×10^(4) L/mol,结合数(n)为0.85,随着温度升高,其Ksv和Ka逐渐降低,表明其淬灭机制为静态淬灭,n值接近1则表明两者可形成1:1复合物。同步荧光光谱表明,胡桃醌与Pin1结合会促使Pin1酪氨酸和色氨酸残基周围微环境疏水性降低,极性增加。圆二色谱揭示胡桃醌与Pin1结合会导致Pin1中的α-螺旋结构减少。热力学参数显示,在293 K条件下,ΔH=12.97 kJ/mol,ΔS=127.83 J/(mol·K),ΔG=-24.49 kJ/mol,表明胡桃醌与Pin1可自发结合,其主要作用力为疏水作用力。分子对接显示,氢键和范德华力在两者作用过程中同样扮演着重要角色。分子对接、定点突变以及分子动力学模拟进一步揭示Pin1催化残基Cys113在胡桃醌结合到Pin1过程中发挥着至关重要的作用。

Juglone is an important active substance in Juglandaceae walnut plant nuts chinense(Jugland mandshurica Maxim)and walnut(Juglans regia L),and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1(Pin1)is a small molecule proteasome that mainly plays a role in signaling transduction.In order to explore the inhibition mechanism of juglone on Pin1,the interaction between Pin1 and juglone was studied by site-directed mutagenesis,computational simulation techniques and multiple spectroscopy.The fluorescence spectra showed that juglone could effectively quench the endogenous fluorescence of Pin1.At 293 K,the quenching constant(Ksv)was 1.36×10^(4) L/mol,the binding constant was(Ka)2.32×10^(4) L/mol,and the binding number(n)was 0.85.With the increase of temperature,Ksv and Ka decreased gradually,which indicated that the quenching mechanism was static quenching,and the binding number n was close to 1,which indicated that they could form 1:1 complex.Synchronous fluorescence spectrum revealed that the combination of juglone with Pin1 could decrease the hydrophobicity and increase the polarity of the microenvironment around the tyrosine and tryptophan residues of Pin1.Circular dichroism revealed that the combination of juglone with Pin1 resulted in the reduction ofα-helical structure in Pin1.Thermodynamic parameters showed thatΔH=12.97 kJ/mol,ΔS=127.83 J/(mol·K),andΔG=-24.49 kJ/mol at 293 K,which indicated that juglone and Pin1 could combine spontaneously,and the main action force was hydrophobic action.Molecular docking showed that hydrogen bonds and van der Waals forces also played important roles in the process.Molecular docking,fluorescence titration and molecular dynamics simulation further revealed that catalytic residue Cys113 of Pin1 played a crucial role in the binding of juglone to Pin1.