Assembly of human C-terminal binding protein (CtBP) into tetramers

Abstract

C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD- and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate- and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer.

CryoEM Structure of CtBP2 Confirms Tetrameric Architecture

“…Although previous studies have proposed dimeric CtBP as the relevant oligomeric state (Nardini et al, 2003;Thio et al, 2004;Nardini et al, 2009;Bi et al, 2018;Mani-Telang et al, 2007;Dcona et al, 2019), our studies with multi-angle light scattering and sitedirected mutagenesis have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers (Bellesis et al, 2018). This was further supported by the observation that CtBP1 and CtBP2 exhibit similar tetrameric assemblies within distinct crystal lattices used for structure determination (Hilbert et al, 2014), resulting in a tetramer model for CtBP.…”

“…Oligomerization is essential for CtBP transcriptional activity, with CtBP forming dimers (Kumar et al, 2002;Nardini et al, 2003) and higher order structures (Bellesis et al, 2018;. Binding of NAD(H) promotes oligomerization of CtBP2, which is required for transcriptional activities (Kumar et al, 2002;Zhang et al, 2002).…”

“…Binding of NAD(H) promotes oligomerization of CtBP2, which is required for transcriptional activities (Kumar et al, 2002;Zhang et al, 2002). In conditions where the level of NAD(H) is low, CtBP2 is mostly dimeric (Bellesis et al, 2018). Increasing the level of NADH in the cell promotes oligomerization of CtBP2.…”

CtBP determines ovarian cancer cell fate through repression of death receptors

“…Of note, previous studies have shown that CtBP polymerization contributes to its regulatory effects, and CtBP dimerization is regulated by cellular NADH binding to its conserved dehydrogenase domain 52,53 . Pharmacologic reduction of NADH depolymerizes CtBP, resulting in induction of certain normally repressed target genes 54,55 . Since CtBP dimers can be disrupted not only by reduction in NADH level, but also by small molecule CtBP dehydrogenase inhibitors 56 , a promising approach for treatment of HGSOC and other CtBP dependent cancers that is currently under investigation may be combining small molecule CtBP inhibition with strategies that reduce cellular NADH level 57 .…”

The C-terminal of the E1A binding protein 2 enhances the metastatic potential of keratinocyte-derived cutaneous squamous carcinoma cells via the gp130/JAK2/Stat1 signaling pathway

“…As it is well acknowledged that tumor cells often exert raised NADH levels owing to NADH production underneath hypoxic and pseudohypoxic environments [13][14][15]. A previous study have implied that NADH can interact CtBP with a high affinity, contribute to a modification in the protein structure that empowers its communication with transcriptional suppressors [16][17][18]. Moreover, it has been suggested that high NADH levels under hypoxic environments lead to an abridged E-cadherin expression level in tumors cells, contribute to the oncogenic role of CtBP in human manligancies [19].…”