基于分子动力学方法研究残基突变对P53构象变化的影响
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摘要
P53作为肿瘤抑制因子，在肿瘤发生、发展及治疗等方面发挥着重要作用。正常P53蛋白具有“基因卫士”的功能，能参与细胞周期阻滞、DNA修复、细胞凋亡等重要的生物学过程。P53蛋白与其它蛋白结合或者发生基因突变，则会导致生物功能的丧失。在所发生的恶性肿瘤中，50%以上存在P53残基突变，而突变主要发生在P53-DNA结合域。人类肿瘤中P53突变主要在高度保守区内，以175、245，248、249、273、282位点突变最高，其中有4个突变热点残基位于高度保守区域IV（残基234-255）。研究残基突变对P53结构的影响，对预防和治疗由残基突变引起的肿瘤疾病具有重要意义。
作为实验研究的有效补充手段，分子动力学模拟在研究生物大分子结构与功能方面具有重要作用。本文利用分子动力学模拟方法，研究P53的R249S、R248W和G245S不同突变对P53肽段构象的影响。选取P53-DNA结合域残基230-258肽段为研究对象，利用GROMACS模拟软件，对野生型肽段wtP53、单点突变型肽段P53-R249S、两点突变型肽段P53-R249S/R248W和三点突变型肽段P53-R249S/R248W/G245S进行分子动力学模拟，每组体系模拟时间为500ns。通过对二级结构、三级结构形成情况，以及构象异质性、结构稳定分析，研究不同残基突变对结构的影响，结果发现：
（1）与wtP53肽段相比，单残基R249S发生突变时，构象虽然仍主要分布在β折叠区，但是其几率减小，同时产生α螺旋构象的倾向增加；肽段两端点残基形成strand几率减小，在突变点附近残基形成strand几率增大；构象自由能较低，结构稳定性较强。单残基R249S突变时，突变点附近结构展开程度及异质性并没有发生较大变化，同时，245和248位点附近结构也没有发生较大变化；单残基R249S突变会使二级结构发生变化，但三级结构近似不变。
（2）R249S和R248W两点同时突变时，将使R249S突变造成的结构变化进一步加剧，其构象虽然仍旧主要分布在β折叠区，但出现α螺旋构象的几率增加；二级结构形成情况变化较大，形成2个turn和3个strand模式，同时三级结构也会发生较大变化。在R249S突变基础上，R248W突变使得突变位点附近结构变得紧凑，肽段结构变得比较稳定，残基涨落较小，结构异质性较低。
（3）在两个残基R249S、R248W突变的基础上，增加第三个残基G245S突变后，R249S和R248W突变对肽段影响减弱，残基主链二面角平均力势分布趋于wtP53的分布；三级结构的形成情况趋于wtP53体系，表明R249S、R248W两突变与G245S突变对肽段结构的影响相反。三残基同时突变导致肽段的结构异质性增大，特别是突变位点245附近残基涨落较大，结构相对松散。
本论文共分五章。第一章为综述，简单介绍了P53蛋白、该蛋白结构、功能以及P53-DNA结合域残基突变。第二章对分子动力学模拟进行了介绍，包括分子动力学模拟原理、分子动力学模拟的应用及发展、常用的GROMACS模拟软件。第三章介绍了本工作的研究对象及方法，包括体系选取、模拟方案以及模拟轨迹分析的几种方法。第四章从不同角度对模拟体系进行了分析研究，在确保模拟收敛的前提下，分析了残基突变对肽段二级结构、三级结构、构象异质性以及结构稳定性的影响。第五章进行了总结和展望。
P53, known as a cancer inhabited factor, plays a key role in tumor occurrence,development and treatment. P53 has“gene guard”function, participating in many biologicalprocesses, such as cell cycle arrest and/or apoptosis and DNA repair. P53 may lose itsfunction when binding with other proteins or appearing gene mutation. In malignant tumor,mutations in P53 are associated with over 50% of human mutations found in tumors,furthermore mainly located in P53-DNA binding domain. In human tumor, P53 mutations arelocated in highly conserved regions, especially 175, 245, 248, 249, 273 and 282 residues aremutated frequently. There are 3“host spots”in region IV (residue 234 -255). The study oneffect of residue mutations on P53 structure was important for the prevention and treatmentthe diseases caused by tumor.
Molecular dynamics simulation, as an effective supplement method to experiment, hasimportant effects on studying structure and function about biological molecules. In this study,the structural characters of the P53 segment were studied by molecular dynamics simulationswith different mutations R249S, R248W and G245S. Four independent simulations forwild-type segment wtP53, one-point mutation segment P53-R249S, two-point mutationsegment R249S/R248W and three-point mutation segment R249S/R248W/G245S whichwere located in the P53-DNA binding domain (from residue 230 to 258) were performed withGROMACS soft package and GROMOS 43A1 force field. Each simulation was lasted for500 ns. The effects of different residue mutations on structure were studied by analyzing theseparameters such as the secondary structure, the tertiary structure, the structure heterogeneityand the structural stability. The results were as follow.
(1) Compared with wtP53 segment, when R249S was mutated, the conformations werestill mainlyβstructures, but the probability decreased and the tendency to produce theα-helix conformation increased. The formation of the strand structures reduced at both ends of thesegment, but near the mutant the probability of forming the strand structures increased. Thefreedom energy of the conformation was lower to cause the structure stably. The structure andthe heterogeneity near the mutation R249S had no changes; meanwhile the structures near theresidues 245 and 248 had no changes. The mutation R249S had effect on the formation ofsecondary structure for some residues, but had little effect on the mode of the ternarystructure.
(2) On the other hand, the R249S/R248W mutation strengthened the effect of R249S onthe segment. The conformation could still mainly keepβstructures, but the probability of theα-helix conformation increased. The formation of the secondary structures which showed 2turns and 3 strands motif, varied considerably; simultaneously a great change of ternarystructure was induced. Based on the mutation R249S, the structure near the mutation becamevery compact and more stable because of R248W. The structure heterogeneity of P53 segmentwhich had the small fluctuation became very low.
(3) The changes of the peptide segment caused by R249S/R248W could decrease withthe mutation G245S. The distribution of potentially mean forces obtained from ramachandranwas similar to wtP53 segment. The formation of secondary structures was similar toP53-R249S segment, but the ternary structure formation was similar to wtP53 segment. Itrevealed that the mutation G245S had an opposite effect on the peptide segmentcorresponding to the mutation R249S/R248W. The structure heterogeneity of P53 segmentchanged very much, especially near the mutation G245S the residues had large fluctuationand the structure was relatively loose.
This paper was divided into 5 chapters. The first chapter was an introduction in which weintroduced P53 protein, the structure and function of calcium protein and the mutation locatedin P53-DNA binding domain. In the second part, molecular dynamics simulation wasintroduced including molecular dynamics simulation principles, the application anddevelopment of molecular dynamics simulation and the GROMACS simulation softwarefrequently used. In the third part, we introduced the research system and methods containingthe system selection, the simulation scheme and several analysis methods of the simulation trajectory. We analyzed the simulation systems in various degrees in the fourth part. At first ,we proved the simulation was ensured convergence. We calculated the secondary structure,the ternary structure, the structure heterogeneity and the structural stability for the differentmutation systems. The summary and prospect were presented in the last chapter.
作为实验研究的有效补充手段，分子动力学模拟在研究生物大分子结构与功能方面具有重要作用。本文利用分子动力学模拟方法，研究P53的R249S、R248W和G245S不同突变对P53肽段构象的影响。选取P53-DNA结合域残基230-258肽段为研究对象，利用GROMACS模拟软件，对野生型肽段wtP53、单点突变型肽段P53-R249S、两点突变型肽段P53-R249S/R248W和三点突变型肽段P53-R249S/R248W/G245S进行分子动力学模拟，每组体系模拟时间为500ns。通过对二级结构、三级结构形成情况，以及构象异质性、结构稳定分析，研究不同残基突变对结构的影响，结果发现：
（1）与wtP53肽段相比，单残基R249S发生突变时，构象虽然仍主要分布在β折叠区，但是其几率减小，同时产生α螺旋构象的倾向增加；肽段两端点残基形成strand几率减小，在突变点附近残基形成strand几率增大；构象自由能较低，结构稳定性较强。单残基R249S突变时，突变点附近结构展开程度及异质性并没有发生较大变化，同时，245和248位点附近结构也没有发生较大变化；单残基R249S突变会使二级结构发生变化，但三级结构近似不变。
（2）R249S和R248W两点同时突变时，将使R249S突变造成的结构变化进一步加剧，其构象虽然仍旧主要分布在β折叠区，但出现α螺旋构象的几率增加；二级结构形成情况变化较大，形成2个turn和3个strand模式，同时三级结构也会发生较大变化。在R249S突变基础上，R248W突变使得突变位点附近结构变得紧凑，肽段结构变得比较稳定，残基涨落较小，结构异质性较低。
（3）在两个残基R249S、R248W突变的基础上，增加第三个残基G245S突变后，R249S和R248W突变对肽段影响减弱，残基主链二面角平均力势分布趋于wtP53的分布；三级结构的形成情况趋于wtP53体系，表明R249S、R248W两突变与G245S突变对肽段结构的影响相反。三残基同时突变导致肽段的结构异质性增大，特别是突变位点245附近残基涨落较大，结构相对松散。
本论文共分五章。第一章为综述，简单介绍了P53蛋白、该蛋白结构、功能以及P53-DNA结合域残基突变。第二章对分子动力学模拟进行了介绍，包括分子动力学模拟原理、分子动力学模拟的应用及发展、常用的GROMACS模拟软件。第三章介绍了本工作的研究对象及方法，包括体系选取、模拟方案以及模拟轨迹分析的几种方法。第四章从不同角度对模拟体系进行了分析研究，在确保模拟收敛的前提下，分析了残基突变对肽段二级结构、三级结构、构象异质性以及结构稳定性的影响。第五章进行了总结和展望。
P53, known as a cancer inhabited factor, plays a key role in tumor occurrence,development and treatment. P53 has“gene guard”function, participating in many biologicalprocesses, such as cell cycle arrest and/or apoptosis and DNA repair. P53 may lose itsfunction when binding with other proteins or appearing gene mutation. In malignant tumor,mutations in P53 are associated with over 50% of human mutations found in tumors,furthermore mainly located in P53-DNA binding domain. In human tumor, P53 mutations arelocated in highly conserved regions, especially 175, 245, 248, 249, 273 and 282 residues aremutated frequently. There are 3“host spots”in region IV (residue 234 -255). The study oneffect of residue mutations on P53 structure was important for the prevention and treatmentthe diseases caused by tumor.
Molecular dynamics simulation, as an effective supplement method to experiment, hasimportant effects on studying structure and function about biological molecules. In this study,the structural characters of the P53 segment were studied by molecular dynamics simulationswith different mutations R249S, R248W and G245S. Four independent simulations forwild-type segment wtP53, one-point mutation segment P53-R249S, two-point mutationsegment R249S/R248W and three-point mutation segment R249S/R248W/G245S whichwere located in the P53-DNA binding domain (from residue 230 to 258) were performed withGROMACS soft package and GROMOS 43A1 force field. Each simulation was lasted for500 ns. The effects of different residue mutations on structure were studied by analyzing theseparameters such as the secondary structure, the tertiary structure, the structure heterogeneityand the structural stability. The results were as follow.
(1) Compared with wtP53 segment, when R249S was mutated, the conformations werestill mainlyβstructures, but the probability decreased and the tendency to produce theα-helix conformation increased. The formation of the strand structures reduced at both ends of thesegment, but near the mutant the probability of forming the strand structures increased. Thefreedom energy of the conformation was lower to cause the structure stably. The structure andthe heterogeneity near the mutation R249S had no changes; meanwhile the structures near theresidues 245 and 248 had no changes. The mutation R249S had effect on the formation ofsecondary structure for some residues, but had little effect on the mode of the ternarystructure.
(2) On the other hand, the R249S/R248W mutation strengthened the effect of R249S onthe segment. The conformation could still mainly keepβstructures, but the probability of theα-helix conformation increased. The formation of the secondary structures which showed 2turns and 3 strands motif, varied considerably; simultaneously a great change of ternarystructure was induced. Based on the mutation R249S, the structure near the mutation becamevery compact and more stable because of R248W. The structure heterogeneity of P53 segmentwhich had the small fluctuation became very low.
(3) The changes of the peptide segment caused by R249S/R248W could decrease withthe mutation G245S. The distribution of potentially mean forces obtained from ramachandranwas similar to wtP53 segment. The formation of secondary structures was similar toP53-R249S segment, but the ternary structure formation was similar to wtP53 segment. Itrevealed that the mutation G245S had an opposite effect on the peptide segmentcorresponding to the mutation R249S/R248W. The structure heterogeneity of P53 segmentchanged very much, especially near the mutation G245S the residues had large fluctuationand the structure was relatively loose.
This paper was divided into 5 chapters. The first chapter was an introduction in which weintroduced P53 protein, the structure and function of calcium protein and the mutation locatedin P53-DNA binding domain. In the second part, molecular dynamics simulation wasintroduced including molecular dynamics simulation principles, the application anddevelopment of molecular dynamics simulation and the GROMACS simulation softwarefrequently used. In the third part, we introduced the research system and methods containingthe system selection, the simulation scheme and several analysis methods of the simulation trajectory. We analyzed the simulation systems in various degrees in the fourth part. At first ,we proved the simulation was ensured convergence. We calculated the secondary structure,the ternary structure, the structure heterogeneity and the structural stability for the differentmutation systems. The summary and prospect were presented in the last chapter.
引文
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