Mechanism of CREB recognition and coactivation by the CREB-regulated transcriptional coactivator CRTC2

Abstract

Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.

Fig. 1.

Assembly of a CRTC2:CREB:CRE ternary complex using relevant interaction domains. (A) SEC-MALS profiles of mixtures of various CRTC2 peptides with a CREB bZip:CRE complex. The profiles of the CREB:CRE complex or just the CRE are shown for comparison. The thick lines within or above each peak correspond to the range of molar masses detected for that species. (B) FA curves from titrations conducted with a fluoresceinated double-stranded oligonucleotide harboring various CREs with a CREB bZip peptide (Left). FA curves from titrations of the respective fluoresceinated CRE-bound CREB bZip conducted with increasing amounts of GST-CRTC2 (residues 1–55) or CRTC2 (1–116).

Fig. 2.

Conserved cysteines in the CREB bZip domain perform contrasting roles in stabilizing the CRTC2 interaction. (A) A multiple sequence alignment of CREB paralogs and orthologs. Species abbreviations: Bt, Bos taurus; Cf, Canis familiaris; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Xl, Xenopus laevis. The symbols at the bottom identify cysteines (diamonds) and residues that engage in intersubunit hydrogen bonding and/or electrostatic interactions in CREB bZip:CRE complex (16) (Protein Data Bank ID: 1DH3). The “a” and “d”positions of the heptad repeat in the leucine zipper are identified above the alignment. (B) Changes in CRE- and CRTC2-binding affinity (Left and Right, respectively) induced by cysteine mutations in the context of different CREs. Changes in CRTC2-binding affinity shown on log scale. (C) Relative activities of wild-type and mutant CREB polypeptides. CRE-luc reporter activity in HEK293T cells expressing Halo-tagged wild-type, C300S, or R314A CREB. Treatment with FSK (10 μM) or DMSO vehicle (4 h) indicated. Relative luciferase activity normalized to β-galactosidase activity from a cotransfected Rous sarcoma virus (RSV)–β-gal plasmid.

Fig. 3.

A highly conserved motif in the CBD of CRTC2 forms a helical structure. (A) Multiple sequence alignment spanning the first 55 residues of CRTC2 orthologs and paralogs. The human, mouse, and rat versions of CRTC1, CRTC2, and CRTC3 sequences are identical in this segment. (B) A 2.0-Å crystal structure of a selenomethionine-labeled CRTC2 CBD peptide (residues 18–50) shown along with the 2F_o-F_c electron density map contoured at 1.5 σ. CRCT2 residues Asn18, Pro19, Gly48, Ser49, and Thr50 are disordered in the crystal with no interpretable density; Arg20 through Ile47 form a single continuous α-helix. Green spheres represent Zn²⁺ ions, presumably from the crystallization buffer.

Fig. 4.

CBD in CRTC2 forms an extensive protein–protein interface with the bZip domain in CREB. (A) Multiresidue alanine scanning mutagenesis of residues 19 through 51 of the CRTC2 CBD. The CREB-binding activity of the mutant proteins generated in the GST-CRTC2 (residues 1–65) background was evaluated by gel mobility shift assays. Crescendo bars indicate increasing concentrations of GST-CRTC2 (0.5, 1, and 2 μM). Filled circle, arrowhead, and star identify the locations of the free probe, CREB bZip-bound CRE, and the CRTC2:CREB:CRE complex on the gels, respectively. CRTC2 residues are highlighted according to the effect of the mutations, with light blue, orange, and red denoting small, moderate, and severe reductions, respectively, on CREB binding. (B) Reductions (log scale) in CREB-binding affinity of various CRTC2 alanine, glutamate, and proline mutants in the context of the somatostatin CRE. (C) Reductions (log scale) in CRE- and CRTC2-binding affinity (Left and Right, respectively) of various CREB mutants.

Fig. 5.

Structural model for the interaction between CRTC2 CBD and CRE-bound CREB bZip. Undocked models of CRE-bound CREB bZip (Left) and CRTC2 (Center), with residues deemed functionally significant by mutagenesis studies colored in magenta and purple, respectively. The cysteine residue (Cys300) in bZip whose mutation to serine enhances the CREB–CRTC2 interaction is shown in cyan. (Right) A manually docked model based on mutational data and the crystal structures of CRTC2 CBD and CREB bZip bound to somatostatin CRE (16) (Protein Data Bank ID: 1DH3).