CryoEM Structure of CtBP2 Confirms Tetrameric Architecture

C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis and metastasis. CtBP proteins are activated under hypoxic conditions where NAD(H) levels tend to be higher. NADH-dependent activation of CtBP2 has direct implication in multiple types of cancers and poor patient prognosis. Central to understanding the transcriptional activation of CtBP in oncogenesis is to uncover how NAD(H) triggers protein assembly, what level of assembly occurs and if the oligomeric form is required for oncogenic transcriptional function. Previous studies have proposed dimeric CtBP as the relevant oligomeric state, however our studies with multi-angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the cryoEM structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is indeed tetrameric. The physiological relevance of tetrameric CtBP2 was tested in HCT116; CtBP2 −/− cells transfected with tetramer destabilizing mutants. Mutants that inhibit tetramer formation are defective for the CtBP2 transcriptional repression of CDH1 (E-cadherin), transcriptional activation of TIAM1 and exhibit a decrease in the ability to promote cell migration, providing the first direct evidence for the role of tetrameric CtBP2 in oncogenesis. Together with our cryoEM studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.