Properties of human centrin 2 and cooperative effects between the N- and C-terminal domains

Abstract

Human centrin 2 (HsCen2) is a Ca²⁺-binding protein belonging to the highly conserved calmodulin superfamily, and it is organized by two domains, namely, N-terminal and C-terminal domains. However, the combined effect between the N- and C-terminal domains of HsCen2 is not clear. The aggregation and the endonuclease-like activities of HsCen2 and their isolated C-terminal and N-terminal domains (C-HsCen2 and N-HsCen2) were investigated by isothermal titration calorimetry (ITC), far-UV CD spectroscopy, DOCK analysis, and agarose gel electrophoresis in 10 mM Hepes, pH 7.4. The results proved the presence of a cooperative effect between the N- and C-terminal domains of HsCen2. The presence of the N-terminal domain facilitated the binding of Tb³⁺ to the C-terminal domain of HsCen2 and enhanced Tb³⁺-induced HsCen2 aggregation. Interestingly, DNA can bind to HsCen2 or C-HsCen2 or N-HsCen2, and the affinity of C-HsCen2 binding to DNA is stronger than that of intact HsCen2 to DNA. In addition, HsCen2, isolated C-HsCen2, and isolated N-HsCen2 exhibited endonuclease-like activities that induced DNA strand break through the hydrolysis pathway, and the endonuclease-like activity was of the following order: N-HsCen2 > C-HsCen2 > HsCen2. The results are extensively discussed, and the cooperative effect between the N- and C-terminal domains of HsCen2 is explored.