当前位置: X-MOL 学术Biopolymers › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV
Biopolymers ( IF 2.9 ) Pub Date : 2020-08-28 , DOI: 10.1002/bip.23396
Sasthi Charan Mandal 1 , Lakshmi Maganti 2 , Manas Mondal 3 , Jaydeb Chakrabarti 1, 4
Affiliation  

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg2+ ions, EcoRV is able to cleave the DNA but not in presence of Ca2+, although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg2+(A)‐EcoRV‐DNA complex compared to Ca2+(S)‐EcoRV‐DNA complex using all‐atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca2+(S)‐EcoRV‐DNA compared to Mg2+(A)‐EcoRV‐DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca2+(S) bound complex than in Mg2+(A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein‐DNA systems.

中文翻译:

通过限制性内切酶 EcoRV 对 DNA 切割中金属离子辅因子特异性的微观洞察

限制性核酸内切酶通过将传入的外源 DNA 切割成片段来保护细菌细胞免受噬菌体感染。在存在 Mg2+ 离子的情况下,EcoRV 能够切割 DNA,但在存在 Ca2+ 时不能切割,尽管蛋白质在两种金属离子存在的情况下都与 DNA 结合。我们尝试使用构象热力学来理解这种差异。我们计算了构象自由能和构象自由度熵的变化,如 DNA 碱基对步骤和 Mg2+(A)-EcoRV-DNA 复合物中蛋白质残基的二面角与 Ca2+(S)-EcoRV-DNA 复合物相比,使用所有-配合物的原子分子动力学(MD)轨迹。我们发现,尽管两种复合物的构象稳定且有序,但在存在两种不同金属离子的情况下,个体的自由度表现不同。与 Mg2+(A)-EcoRV-DNA 相比,Ca2+(S)-EcoRV-DNA 裂解区的碱基对高度无序。与切割位点附近的金属离子配位的酸性残基之一 ASP90 构象不稳定和无序,而碱性残基 LYS92 在 Ca2+(S) 结合复合物中比在 Mg2+(A) 结合复合物中获得构象稳定性和有序性. 增强的波动阻碍了金属离子在 DNA 的易裂解磷酸盐附近的位置。通过替换金属离子观察到切割区域中构象稳定性和秩序的类似丧失。考虑到将金属离子置于易裂解磷酸盐附近作为裂解作用的要求,我们的结果表明,碱基对步骤和蛋白质残基的构象稳定性和顺序的变化导致酶的辅因子敏感性。我们基于微观构象变量波动的方法可用于了解其他蛋白质-DNA 系统中的酶活性。
更新日期:2020-08-28
down
wechat
bug