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. 2012 Dec 7;287(50):41914-21.
doi: 10.1074/jbc.M112.418855. Epub 2012 Oct 18.

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

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Free PMC article

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

Qiang Ge et al. J Biol Chem. .
Free PMC article

Abstract

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 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well defined α-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-binding modes. ChREBP α2 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 α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β·ChREBP α2 complex.

Figures

FIGURE 1.
FIGURE 1.
Domain organization and the sequence of the N-terminal glucose-responsive regulatory region of ChREBP. A, residues 81–196 of the N-terminal regulatory region, including the predicted α1, α2, and α3 helices, the nuclear export signal 2 (NES2), nuclear localization signal (NLS), and Ser-196 phosphorylation site, were found in a complex with 14-3-3β. B, SDS-PAGE analysis of 14-3-3β·ChREBP crystals revealed the presence of both 14-3-3β and ChREBP fragment. C, simulated annealing omit map of ChREBP α2 helix bound to 14-3-3β and the interface SO4 molecule. The maps are contoured at 1.0σ for ChREBP and 2.0σ for the sulfate.
FIGURE 2.
FIGURE 2.
Overall structure of 14-3-3β·ChREBP complex. A, ribbon diagram of a monomer of the 14-3-3β (cyan) complexed with the α2 helix of ChREBP (orange). The SO4 molecule is shown as sticks. B, two orthogonal views of 14-3-3β dimer in complex to ChREBP α2.
FIGURE 3.
FIGURE 3.
Involvement of a free sulfate at the interface between 14-3-3β and ChREBP α2. A, detailed interactions between 14-3-3β (cyan) and ChREBP α2 (orange). Residues involved in the intermolecular contacts are shown as sticks. A water molecule (WAT) is shown as a small red sphere. Hydrogen bonds are shown as dotted lines. B, comparison of the 14-3-3β·ChREBP interface with that between 14-3-3ξ and a phosphorylated histone H3 peptide (pH 3, in green). Residues of 14-3-3ξ are labeled.
FIGURE 4.
FIGURE 4.
Mutagenesis and binding analysis of ChREBP and 14-3-3β. A, pull-down assays were performed to assess the binding between WT and mutant ChREBP and endogenous 14-3-3 proteins. Full-length FLAG-tagged ChREBP proteins were expressed in HEK293T cells and purified via the anti-FLAG resin. The association of 14-3-3 proteins with ChREBP was detected via Western blotting using anti-14-3-3 pan-antibody. B, pull-down assays of the binding between the WT ChREBP and exogenously expressed WT and mutant 14-3-3β. C, ITC experiments were performed to measure the binding constant between a ChREBP peptide containing the α2 helix (residues 117–140) to the WT and two 14-3-3β mutants, R62A and E182A. The isotherm titrations from three data sets were analyzed with ORIGIN version 7.0 software package (MicroCal/GE Healthcare). They correspond to wild-type (black squares), R62A (black circles), E182A (black triangles), respectively.
FIGURE 5.
FIGURE 5.
Comparison of 14-3-3β·ChREBP with 14-3-3β·ExoS revealed two distinct nonphosphorylated peptide-binding modes. A, two orthogonal views of the superposition of 14-3-3β (cyan)·ChREBP (blue) complex with 14-3-3β (pink)·ExoS (magenta) complex. The apo-14-3-3β (yellow, Protein Data Bank code 2BQ0) in closed conformation is also superimposed. The sulfate in the complex of 14-3-3β·ChREBP is shown as sticks. B, ExoS peptide and ChREBP α2 bind to the nonoverlapping sites at the conserved peptide-binding groove of 14-3-3β. The molecular surface of 14-3-3β is shown and colored by electrostatic potentials. Negative potential is colored red and positive potential blue.

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