Purification And Nuclear Magnetic Resonance Study Of Mutant Bcl-2(mimetic Phosphorylation)

Significance of multi-site phosphorylation of Bcl-2 protein within the flexible loop domain is still under debate,which is partly due to the absence of structural biology study of phosphorylated Bcl-2(pBcl-2).Furthermore,p Bcl-2 has been identified as a resistant of the first FDA approved Bcl-2 inhibitor venetoclax(ABT-199),and its analogues ABT-737 and ABT-263 in a broad spectrum of cancers.Structural study of p Bcl-2 would improve our understanding of how p Bcl-2 regulates apoptosis and how it resists available Bcl-2 inhibitors designed based on the structure of BH3 groove on Bcl-2 surface.Unfortunately,such studies have been hampered by difficulties in obtaining sufficient quantities of Bcl-2 protein because of the severe solubility problems.Previous work about Bcl-2 protein were carried out using structurally modified Bcl-2/Bcl-x L chimeras.However the phosphorylated sites t69,s70,and s87 were then ignored.Therefore,we constructed a phosphomimetic mutant Bcl-2(2-206)(t69e,s70 e and s87e)(EEE-Bcl-2(2-206))by replacing the negatively charged phosphoserine and phosphothreonine with glutamic acid and deleting a short C-terminal sequence to improve solubility.We further constructed a phosphomimetic mutant Bcl-2(62-206)(t69e,s70 e and s87e)(EEE-Bcl-2-EK)the BH4 and part of loop region was trunked(residues 2-61)to produce soluble and stable protein in large scale to obtain a well dispersed 15N-1H heteronuclear single quantum coherence(HSQC)NMR spectroscopy,while the three phosphorylation sites were remained and mutated to glutamate.The recombinant mutant EEE-Bcl-2-EK closely mimics the biochemically and functionally characterized of the native phosphorylated Bcl-2 which is differ from that of Bcl-2 protein.The difference between BH3 groove of EEE-Bcl-2-EK and Bcl-2 protein in two-dimensional NMR spectra demonstrated the phosphorylation in loop region induced structural alternation in active domain accounts for the phosphorylation regulated anti-apoptotic function of Bcl-2.