Expression and Solution NMR Study of Multi-site Phosphomimetic Mutant BCL-2 Protein

Author(s): Ting Song, Keke Cao, Yu dan Fan, Zhichao Zhang*, Zong W. Guo, Min H. Zhang, Peng Liu.

Journal Name: Protein & Peptide Letters

Volume 26 , Issue 6 , 2019

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Graphical Abstract:


Background: 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.

Objective: The purpose of the study is to explore the phosphorylation induced structural changes of BCL-2 protein.

Methods: We constructed a phosphomietic mutant BCL-2(62-206) (t69e, s70e and s87e) (EEEBCL- 2-EK (62-206)), in which the BH4 domain and the part of loop region was truncated (residues 2-61) to enable a backbone resonance assignment. The phosphorylation-induced structural change was visualized by overlapping a well dispersed 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy between EEE-BCL-2-EK (62-206) and BCL-2.

Results: The EEE-BCL-2-EK (62-206) protein reproduced the biochemical and cellular activity of the native phosphorylated BCL-2 (pBCL-2), which was distinct from non-phosphorylated BCL-2 (npBCL-2) protein. Some residues in BH3 binding groove occurred chemical shift in the EEEBCL- 2-EK (62-206) spectrum, indicating that the phosphorylation in the loop region induces a structural change of active site.

Conclusion: The phosphorylation of BCL-2 induced structural change in BH3 binding groove.

Keywords: Phosphorylated BCL-2, purification, NMR spectroscopy, structural change, chemical shift, BH3 binding groove.

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Article Details

Year: 2019
Page: [449 - 457]
Pages: 9
DOI: 10.2174/0929866526666190327121225
Price: $65

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