Structural Characterization of a Unique Interface between Carbohydrate Response Element-binding Protein (ChREBP) and 14-3-3β Protein

Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3 beta bound to the N-terminal regulatory region of ChREBP at 2.4 angstrom resolution. The crystal structure revealed that the alpha 2 helix of ChREBP (residues 117-137) adopts a well defined alpha-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-bindingmodes. ChREBP alpha 2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP alpha 2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3 beta. ChREBP alpha 2 complex.