Study On The Structure Of Interaction Between Copper,zinc Superoxide Dismutase And DNA

Cu/Zn superoxide dismutase(SOD1)is an important antioxidant enzyme widely distributed in the cell.Copper ion is the active center,catalyzes the disproportionation of superoxide anion(O2·-)to hydrogen peroxide(H2O2)and O2,and maintains the homeostasis of reactive oxygen species(ROS).The previous studies found that DNA could bind to SOD1,and three SOD1 binding DNA sequences were obtained based on protein microarray in vitro.With the concentrations of H2O2 increasing,cytosolic SOD1 was phosphorylated and transported to the nucleus,regulate the expression of up to 123 genes.Our group used ChIP-Seq assay to obtain the specific DNA sequence named S1 which could bind to SOD1,and verified that SOD1 and S1 formed a complex using EMSA assay and measured the binding constant.In addition,some of the properties and structural features of SOD1 are similar to metalloregulatory proteins such as PerR,SoxR and MarR in microbial cells.Therefore,we hypothesized that SOD1 may be a metal regulatory protein that binds to DNA and regulates gene expression.In this work,first of all,we explored the influence of a series of chelators on the structure and activity of SOD1.The results indicated that DS14 could efficiently inhibit SOD1 activity both in solution and in the cell,and affected the secondary structure of SOD 1.Secondly,this inhibitor could also regulate intracellular ROS levels by SOD1 inhibition.Finally,Autodock was performed to speculate the possible mechanism of inhibition.Furthermore,we used HADDOCK to explore the possible binding mode of SOD1 and specific DNA sequence,and confirmed the binding mode by small angle X-ray scattering.The results showed that the conformation of SOD I was closer after binding to DNA,and the binding mode was consistent with the docking results.Moreover,it was found that the conformation of complex treated with copper chelators were similar to the one under oxidized condition.The simulated structure of complex was looser and DNA dissociated from SOD I,indicating that copper ion played an important role in the binding of SOD1 and DNA,however,the reducing conditions had no effect on the conformation of complex.In addition,we optimized the crystallization conditions of SOD1 complexes mainly by means of vapor diffusion.Some crystals with good diffraction quality were obtained,but no expected results were achieved after analysis.However,we summarized the culture methods and conditions of the complex crystals,which laid a foundation for the subsequent work.In summary,DS14,could inhibit SOD 1 activity efficiently and affect protein structure.The binding mode of SOD1 and DNA in solution was analyzed,by studying the effects of redox conditions and metal chelators on the conformation of complex in solution.It was shown that either copper ions being chelated or coordinated amino acids being oxidized would cause dissociation of SOD1 and DNA.