Expression and solution NMR study of multi-site phosphomimetic mutant BCL-2 protein.

The significance of multi-site phosphorylation of BCL-2 protein in the flexible loop domain remains controversial, in part due to the lack of structural biology studies of phosphorylated BCL-2. We constructed a phosphomietic mutant BCL-2(62-206) (t69e, s70e and s87e) (EEE-BCL-2-EK(62-206)), in which the BH4 domain and the part of loop region was trunkated (residues 2-61). In this way,we obtained a mass of soluble and stable phosphomimetic proteins to produce a well dispersed 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The mutant protein reproduced the biochemical and cellular activity of the native phosphorylated BCL-2 (pBCL-2), which was distinct from nonphosphorylated BCL-2 (npBCL-2) protein. The difference between BH3 groove of EEE-BCL-2-EK(62-206) and BCL-2 protein in two-dimensional NMR spectra demonstrated that the phosphorylation induced structural change in active domain.This explains that phosphorylation regulates the anti-apoptotic function of BCL-2.