Department of Biochemistry and Molecular Pharmacology; Schiffer Lab
Amino Acids, Peptides, and Proteins | Biochemistry | Enzymes and Coenzymes | Medicinal and Pharmaceutical Chemistry | Medicinal-Pharmaceutical Chemistry | Neoplasms | Nucleic Acids, Nucleotides, and Nucleosides | Structural Biology
C-terminal binding proteins (CtBPs) are co-transcriptional factors that play key roles in cell fate. We have previously shown that NAD(H) promotes the assembly of similar tetramers from either human CtBP1 and CtBP2 and that CtBP2 tetramer destabilizing mutants are defective for oncogenic activity. To assist structure-based design efforts for compounds that disrupt CtBP tetramerization, it is essential to understand how NAD(H) triggers tetramer assembly. Here, we investigate the moieties within NAD(H) that are responsible for triggering tetramer formation. Using multi-angle light scattering (MALS) we show that ADP is able to promote tetramer formation of both CtBP1 and CtBP2, whereas AMP promotes tetramer assembly of CtBP1, but not CtBP2. Other NAD(H) moieties that lack the adenosine phosphate, including adenosine and those incorporating nicotinamide, all fail to promote tetramer assembly. Our crystal structures of CtBP1 with AMP reveal participation of the adenosine phosphate in the tetrameric interface, pinpointing its central role in NAD(H) linked assembly. CtBP1 and CtBP2 have overlapping but unique roles, suggesting that a detailed understanding of their unique structural properties might have utility in the design of paralog specific inhibitors. We investigated the different responses to AMP through a series of site-directed mutants at 13 positions. These mutations reveal a central role for a hinge segment, which we term the 120s hinge, that connects the substrate with coenzyme binding domains and influences nucleotide binding and tetramer assembly. Our results provide insight into suitable pockets to explore in structure-based drug design to interfere with co-transcriptional activity of CtBP in cancer.
Cancer target, CtBP MALS, NAD(H), crystallography, dehydrogenase, structural biology, tetrameric assembly, transcription coregulator
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© 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
DOI of Published Version
Nichols JC, Schiffer CA, Royer WE Jr. NAD(H) phosphates mediate tetramer assembly of human C-terminal binding protein (CtBP). J Biol Chem. 2021 Jan 29:100351. doi: 10.1016/j.jbc.2021.100351. Epub ahead of print. PMID: 33524397. Link to article on publisher's site
The Journal of biological chemistry
Nichols JC, Schiffer CA, Royer WE. (2021). NAD(H) phosphates mediate tetramer assembly of human C-terminal binding protein (CtBP). University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1016/j.jbc.2021.100351. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1919
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