6zwk | pdb_00006zwk
Crystal structure of the phosphorylated C-terminal tail of histone H2AX in complex with a specific nanobody (C6 gammaXbody)
Structural highlights
FunctionH2AX_HUMAN Variant histone H2A which replaces conventional H2A in a subset of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. Required for checkpoint-mediated arrest of cell cycle progression in response to low doses of ionizing radiation and for efficient repair of DNA double strand breaks (DSBs) specifically when modified by C-terminal phosphorylation.[1] [2] [3] [4] Publication Abstract from PubMedHistone H2AX phosphorylated at serine 139 (gamma-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While gamma-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to gamma-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of gamma-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize gamma-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect gamma-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision gamma-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions. A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.,Moeglin E, Desplancq D, Stoessel A, Massute C, Ranniger J, McEwen AG, Zeder-Lutz G, Oulad-Abdelghani M, Chiper M, Lafaye P, Di Ventura B, Didier P, Poterszman A, Weiss E Cancers (Basel). 2021 Jul 1;13(13):3317. doi: 10.3390/cancers13133317. PMID:34282773[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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