Crystal Structure of the CCAAT Box/Enhancer-binding Protein β Activating Transcription Factor-4 Basic Leucine Zipper Heterodimer in the Absence of DNA

Abstract

The crystal structure of the heterodimer formed by the basic leucine zipper (bZIP) domains of activating transcription factor-4 (ATF4) and CCAAT box/enhancerbinding protein ␤ (C/EBP␤), from two different bZIP transcription factor families, has been determined and refined to 2.6 Å. The structure shows that the heterodimer forms an asymmetric coiled-coil. Even in the absence of DNA, the basic region of ATF4 forms a continuous ␣-helix, but the basic region of C/EBP␤ is disordered. Proteolysis, electrophoretic mobility shift assay, circular dichroism, and NMR analyses indicated that (i) the bZIP domain of ATF4 is a disordered monomer and forms a homodimer upon binding to the DNA target; (ii) the bZIP domain of ATF4 forms a heterodimer with the bZIP domain of C/EBP␤ that binds the cAMP response element, but not CCAAT box DNA, with high affinity; and (iii) the basic region of ATF4 has a higher ␣-helical propensity than that of C/EBP␤. These results suggest that the degree of ordering of the basic region and the fork and the dimerization properties of the leucine zipper combine to distinguish the structurally similar bZIP domains of ATF4 and C/EBP␤ with respect to DNA target sequence. This study provides insight into the mechanism by which dimeric bZIP transcription factors discriminate between closely related but distinct DNA targets.Protein-protein interactions are involved in a number of different cellular processes, including the regulation of transcription. Many, if not most, transcription factors, including the bZIP 1 proteins, bind their cognate DNA elements as dimers. Dimerization between transcription factors from the same or different families can generate considerable functional diversity using only a relatively small number of components. By binding with different specificity to distinct DNA sites within promoters, heterodimeric transcription factors facilitate the positioning and orientation of proteins on DNA, thus providing distinct surfaces for interaction with other transcriptional regulatory proteins bound to adjacent DNA sites. Among coiled-coil proteins, bZIP family members form dimers via their characteristic leucine zipper helices, which consist of seven-residue (heptad) repeats, (abcdefg) n . Dimer formation by bZIP proteins is selective: some form only homodimers, some form only heterodimers, and others form both homo-and heterodimers. Hydrophobic interactions by leucine and other hydrophobic amino acids in positions 3 and 4 in the helix (the a and d positions of the helical wheel) form the hydrophobic core of the bZIP dimer. Leucine residues occupy most of the d positions in the leucine zipper helix. At the same time, hydrophilic amino acids located just outside of the hydrophobic core (the e and g positions of the helical wheel) also participate in stabilizing the dimer. A highly conserved asparagine amino acid in the a position in the middle of the leucine zipper helix has been shown to destabilize oligomer formation in several bZIP dimers (1). Thus, depending on the precise distribution of h...

NUTRITIONAL CONTROL OF GENE EXPRESSION: How Mammalian Cells Respond to Amino Acid Limitation

“…Selected ATF members are known to heterodimerize within the ATF family as well as with members of another bZIP subgroup, the C/EBP family. In particular, a C/EBPβ-ATF4 complex has been detected at cAMP response elements (114) and the crystal structure of the C/EBPβ-ATF4 heterodimer has been obtained (95). As described below, the human CHOP promoter contains a C/EBP-ATF composite site (123), at which Fawcett et al (30) observed by electrophoresis mobility shift analysis (EMSA) a transient ATF4 binding 2 hours after initiating arsenite-induced stress.…”

Multifarious Roles of Intrinsic Disorder in Proteins Illustrate Its Broad Impact on Plant Biology

“…Both sequence and experimental analyses have shown that the Leucine zipper regions always exist in a highly ordered a-helix, while the basic regions, in the absence of DNA, populate an ensemble of highly dynamic transient structures with substantial helical character (Bracken et al, 1999). The basic regions can be completely structured (e.g., ATF4; Podust et al, 2001), contain a certain amount of helical secondary structure (e.g., HY5; Yoon et al, 2006), or be completely disordered (e.g., Opaque-2; Moreau et al, 2004). Experimental evidence has suggested that, in complex with DNA, the basic regions uniformly form a-helical conformations (Hollenbeck et al, 2002).…”

GADD45β Enhances Col10a1 Transcription via the MTK1/MKK3/6/p38 Axis and Activation of C/EBPβ-TAD4 in Terminally Differentiating Chondrocytes

“…Transactivation domains (TAD1, -2, -3, -4), also known as activation domain modules (ADM1, -2, and -3 for TAD2, -3, and -4, respectively), and repression domains (RD1 and -2) are represented as gray boxes and dashed boxes, respectively. Basic region (BR) and leucine zipper region (LR) are indicated (57). Reporter assays were carried out in ATDC5 cells co-transfected with 300 ng of pRL-TK containing the Gal4 binding site and 50 ng of the GAL4-fused mC/EBP␤ constructs, alone or in combination with 25 ng of GADD45␤ expression vector.…”