Properties Of Human Centrin 2 And Its Regulation By Phosphorylation

The reversible phosphorylation of proteins is a ubiquitous regulatory mechanism in prokaryotes and eukaryotes.In vivo,the phosphorylation of protein plays an important role in various cellular processes.This article uses HsCen2 as the research object to explore the properties and phosphorylation regulation of HsCen2.The research in this article is summarized as follows:Human Centrin 2（HsCen2）consists of two globular lobes:N-terminal and C-terminal domains,which are connected by a flexible linker.Firstly,HsCen2（1-172 aa）,N-HsCen2（1-100 aa）,and C-HsCen2（82-172 aa）were constructed by molecular biological methods.The interaction of HsCen2 with Tb³⁺or DNA or XPC,the aggregation function,and the endonuclease-like activity of HsCen2 were studied by Tb³⁺-fluorescence,isothermal titration calorimetry（ITC）,circular dichroism（CD）spectroscopy,molecular docking simulation（DOCK）,and gel electrophoresis in 10m M Hepes at p H 7.4.HsCen2,C-HsCen2,and N-HsCen2 can bind two Tb³⁺,and the average binding constants were（1.10±0.04）×10⁵ M^-1,（6.36±0.36）×10⁴ M^-1 and（8.78±0.31）×10² M^-1,respectively;The binding ability of HsCen2 and C-HsCen2 to XPC was（1.26±0.32）×10⁸ M^-1 and（3.89±0.64）×10⁷ M^-1,respectively,while the N-terminal does not participate in XPC binding;HsCen2,C-HsCen2,and N-HsCen2can bind to a molecule of DNA,and the binding constants were（7.78±0.03）×10³ M^-1,（1.27±0.28）×10⁴ M^-1 and（1.25±0.17）×10³ M^-1,respectively;The binding of Tb³⁺can induce the aggregation of HsCen2 or C-HsCen2 or N-HsCen2,and the aggregation ability of C-HsCen2 and N-HsCen2 is weaker than that of HsCen2;HsCen2,C-HsCen2,and N-HsCen2 showed endonuclease-like activity,and the molar hydrolysis rate constants for DNA cleavage were 0.62×10³ h^-1·M^-1,3.62×10³ h^-1·M^-1 and 33.85×10³ h^-1·M^-1,respectively.It shows that there exists a certain synergistic effect between the N-and C-domains of HsCen2.Secondly,³¹P-NMR and nondenatured polyacrylamide gel electrophoresis（native PAGE）experiments were used to explore the conditions and influencing factors of HsCen2 phosphorylation.It was proved that protein kinase A（PKA）can phosphorylate residues at Ser170 of HsCen2,and the optimal conditions for phosphorylation of HsCen2 and C-HsCen2 were given.The phosphorylation rate constants for HsCen2 and C-HsCen2 were 0.61±0.13μM·min^-1 and 2.46±0.34μM·min^-1,respectively.It was also found that Ca²⁺,Tb³⁺,XPC,and CPZ could significantly inhibit protein phosphorylation.Subsequently,the properties and functions of phosphorylated HsCen2（HsCen2p）and phosphorylated C-HsCen2（C-HsCen2p）were investigated using the same physicochemical methods.After phosphorylation,both HsCen2p and C-HsCen2p can bind two Tb³⁺,the average binding constants were（3.10±0.21）×10⁴ M^-1 and（8.34±0.29）×10⁴ M^-1,respectively;The binding affinity of XPC to HsCen2p and C-HsCen2p was（7.76±0.32）×10³ M^-1 and（1.65±0.68）×10⁷ M^-1,respectively;Both HsCen2p and C-HsCen2p can form 1:1 complex with DNA,the binding constants were（3.16±0.88）×10⁴ M^-1 and（1.51±0.71）×10⁵ M^-1,respectively;Phosphorylation makes the aggregation of C-HsCen2 induced by Tb³⁺stronger than that of HsCen2;The endonuclease like activity of HsCen2 is stronger than that of C-HsCen2,and the rate constants of DNA hydrolysis by HsCen2p and C-HsCen2p were（90.0±0.24）×10³ h^-1·M^-1 and（300.0±0.01）×10³ h^-1·M^-1,respectively.Finally,the regulation of phosphorylation on the properties and functions of HsCen2 was analyzed.Phosphorylation down-regulates the binding of Tb³⁺to HsCen2;The binding of DNA to HsCen2 was up-regulated;Phosphorylation down-regulates the interaction between HsCen2 and XPC or Kar1p or Rad4,but does not affect the interaction between HsCen2 and Sfi1;Phosphorylation down-regulates the aggregation of HsCen2 by Tb³⁺induced;The endonuclease-like activity of HsCen2 was up-regulated;In the presence of XPC,phosphorylation changes the protein’s cleavage of DNA from double-stranded to single-stranded.