High Resolution NMR Solution Structure of the Leucine Zipper Domain of the c-Jun Homodimer

Abstract

The solution structure of the c-Jun leucine zipper domain has been determined to high resolution using a new calculation protocol designed to handle highly ambiguous sets of interproton distance restraints. The domain comprises a coiled coil of parallel ␣-helices in which most of the hydrophobic residues are buried at the highly symmetrical dimer interface; this interface extends over 10 helical turns and is the most elongated protein domain solved to date using NMR methods. The backbone fold is very similar to that seen in crystal structures of the GCN4 and Jun-Fos leucine zippers; however, in contrast with these crystal structures, the Jun leucine zipper dimer appears to be devoid of favorable intermolecular electrostatic interactions. A polar asparagine residue, located at the dimer interface, forms the sole point of asymmetry in the structure; furthermore, the side chain of this residue is disordered due to motional averaging. This residue, which is highly conserved in the leucine zipper family of transcription factors, provides a destabilizing influence that is likely to facilitate the rapid exchange of zipper strands in vivo.c-Jun is a transcriptional activator that binds to specific DNA sites either as a homodimer or as a heterodimer with the c-Fos protein (1). Together, these proteins play a pivotal role in the regulation of cell growth and differentiation; their role in transducing afferent growth signals into specific genetic responses means that they represent a critical nexus between normal and uncontrolled cell growth. It is therefore not surprising that deregulated expression of both proteins has been implicated in the oncogenic transformation of cells (1). c-Jun is a member of the basic region leucine zipper (bZIP) 1 family of transcription factors, all of which bind to DNA as either homo-or heterodimers (2). Dimerization of bZIP proteins, which is mediated by their leucine zipper domains, is a prerequisite for binding to their cognate DNA enhancer elements (3); dimerization enables the ␣-helical DNA-binding basic domains to be inserted into adjacent major grooves of the dyad symmetric DNA recognition site. Thus, the activity of these proteins is regulated not only by interactions between the protein and DNA, but equally by protein-protein interactions between the leucine zipper domains. There has been considerable activity directed toward understanding the molecular interactions that govern the specificity, affinity, and kinetics of leucine zipper formation. Structural information of the type provided by NMR or x-ray crystallography not only provides a molecular framework for understanding the way in which leucine zippers mediate gene transcription, but also paves the way for rational engineering of dominant-negative leucine zippers or other molecules that might block the activity of bZIP oncoproteins (4). In this study, we have used NMR spectroscopy to determine the solution structure of the c-Jun leucine zipper in order to better understand the interactions that control the affinity and kinetic...

Self-association of the α Subunit of Phosphorylase Kinase as Determined by Two-hybrid Screening

“…Although leucine zippers were first discovered in transcription factors, subsequently they have been shown to mediate dimerization in many non-DNA-binding proteins (28,(35)(36)(37). High resolution NMR and x-ray diffraction studies have shown that zippers interact as parallel coiled-coil ␣-helices, with the dimer interface being formed from the hydrophobic side chains of the a and d positions of one helix packing side by side against the aЈ and dЈ positions, respectively, of the other helix (38). Generally, a conserved Asn residue is buried in the hydrophobic dimer interface and is thought to contribute, via destabilization of the coiled-coil, to the selectivity for hetero-versus homodimerization of monomers (Ref.…”

“…1). Stabilization of leucine zippers is often further mediated via electrostatic interactions between the e-gЈ and eЈ-g residues, respectively (38), and in the PhK leucine zipper, there are several charged residues in these positions available for salt bridge formation (Fig. 1).…”

“…Generally, a conserved Asn residue is buried in the hydrophobic dimer interface and is thought to contribute, via destabilization of the coiled-coil, to the selectivity for hetero-versus homodimerization of monomers (Ref. 38 and references therein). The leucine zipper of PhK aligns well with other known zippers, although it does not contain the conserved Asn but instead has a Cys residue at that position (Fig.…”

Enterophilins, a New Family of Leucine Zipper Proteins Bearing a B30.2 Domain and Associated with Enterocyte Differentiation

“…Indeed, sequence analysis of enterophilin-1 using "Prosite" allows us to predict two coiled coil regions corresponding to residues 10 -15 and 126 -334. However, only enterophilin-1 displays neutral or hydrophobic residues at position d of the ␣ helix, which contributes to the interface between dimerized helices (60,61). It thus remains to be determined whether all enterophilins display any ability to dimerize by discriminating between zipper and non-zipper structures (62).…”

Dimerization via Tandem Leucine Zippers Is Essential for the Activation of the Mitogen-activated Protein Kinase Kinase Kinase, MLK-3

“…X-ray diffraction and NMR spectroscopy studies have demonstrated that leucine zippers form parallel coiledcoil ␣-helices, which wrap around one another via the hydrophobic interaction of the leucine side chains (22,23). Leucine zipper motifs were initially identified in transcription factors and shown to mediate homo-or heterodimerization and play a role in DNA binding (21).…”