Bacterial expression and characterization of the CREB bZip module: Circular dichroism and 2D ¹H‐NMR studies

Abstract

In this paper we describe the expression and purification from bacteria of the recombinant basic leucine zipper (bZip) domain of the cAMP response element binding protein, CREB327. The bZip peptide, CREB259–327, purified to near homogeneity, maintains the sequence‐specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB259–327 was employed to prevent aggregation of the peptide due to cysteine oxidation. The Kd of the purified native and modified CREB259–327 for the CRE site was determined by gel retardation assays to be on the order of 10−7 M. We employed CD spectroscopy to study the folding properties of the native and modified CREB259–327. The CD analyses of the native/modified CREB259–327 peptide demonstrated a 20% increase in the α‐helical content upon binding to the cAMP response‐element. Only a 5% increase in the α‐helical content of CREB259–327 is observed upon binding to the AP‐1 site. This observation contrasts with CREB from the GCN4 protein (Weiss, M.A., et al., 1990, Nature 347, 575–578). In addition, the two‐dimensional (2D) 1H‐NMR studies of the bZip CREB peptide further support the distinct features of the CREB protein, in comparison to GCN4. Analysis by CD and 2D NMR of the dimerization domain of CREB suggests that the distinct DNA binding characteristics of CREB reside in the basic portion of the bZip module.

DNA Binding and Phosphorylation Induce Conformational Alterations in the Kinase-inducible Domain of CREB

“…4A). Only two distinct regions are predicted to be predominantly unfolded, mapping to KID and bZIP, consistent with reports that both the KID and bZIP regions have induced structure upon binding to KIX and DNA, respectively (13,17,18,20,22).…”

“…Together, our data support the existence of three distinct CREB structures: (i) pCREB free in solution, (ii) CREB bound to DNA, and (iii) pCREB bound to DNA. These data are unexpected, as previously published studies have suggested that the only significant structural alterations in the otherwise unstructured protein are induced solely within the bZIP domain upon DNA binding and within the KID region upon KIX binding (13,17,18,20,22).…”

“…Deletion of basic amino acids in KID mimics the phosphorylation-induced conformational alteration, suggesting that intramolecular interactions promote a more compact structure in CREB that are disrupted upon phosphorylation. These data are unexpected, as previously published studies suggest that the only significant structural alterations in the otherwise unstructured protein are induced solely within the bZIP domain and KID upon binding to their respective ligands (13,17,18,20,22). Together, our data show that the KID region has the ability to adopt two unique, DNA-induced conformations that are differentially recognized by opposing regulators.…”

Purification of CREB to apparent homogeneity: Removal of truncation products and contaminating nucleic acid

“…1B, lanes 4, 5). Although it is unusual to heat-purify a protein, many studies have demonstrated that CREB retains activity following heat treatment [2,7,[13][14][15]. This unique property has been attributed to a lack of significant structure in CREB, which is believed to be partially unfolded in solution [8,16].…”

“…Previously published studies have shown that the CREB bZIP domain undergoes a conformational change upon DNA binding [8,14]. To further complement the previous activity assays, we performed limited proteolysis to compare the conformation of CREB purified by the two methods.…”