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.
Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Gene Expression Regulation , Transcription Factors/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Conserved Sequence , Crystallography, X-Ray/methods , Cyclic AMP/chemistry , Cysteine/chemistry , DNA/chemistry , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Leucine Zippers , Molecular Sequence Data , Mutation , Protein Binding , Protein Interaction Mapping , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal Transduction , Transcriptional ActivationABSTRACT
A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, NaV1.2. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel purification protocol has been designed to purify the fusion proteins using metal affinity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome b5 based fusion system for the production of peptides and small proteins (<15 kDa).