Analysis of CYS3 regulator function in Neurospora crassa by modification of leucine zipper dimerization specificity.

The CYS3 positive regulator is a basic region-leucine zipper (bZIP) DNA-binding protein that is essential for the expression of sulfur-controlled structural genes in Neurospora crassa. An approach of modifying the dimerization specificity of the CYS3 leucine zipper was used to determine whether the in vivo regulatory function of CYS3 requires the formation of homodimeric or heterodimeric complexes. Two altered versions of CYS3 with coiled coil elecrostatic interactions favorable to heterodimerization showed restoration of wild-type CYS3 function only when simultaneously expressed in a delta cys-3 strain. In addition, constructs having the CYS3 leucine zipper swapped for that of the oncoprotein Jun or the CYS3 leucine zipper extended by a heptad repeat showed wild-type CYS3 function when transformed into a delta cys-3 strain. Gel mobility shift and immunoprecipitation assays were used to confirm the modified CYS3 proteins dimerization and DNA binding properties. The studies, which precluded wild-type CYS3 dimerization, indicate that in vivo CYS3 is fully functional as a homodimer since no interaction was required with other leucine zipper proteins to activate sulfur regulatory and structural gene expression. The results demonstrate the utility of leucine zipper modification to study the in vivo function of bZIP proteins.     

Attractive interhelical electrostatic interactions in the proline- and acidic-rich region (PAR) leucine zipper subfamily preclude heterodimerization with other basic leucine zipper subfamilies

Basic region-leucine zipper (B-ZIP) proteins homo- or heterodimerize to bind sequence-specific double-stranded DNA. We present circular dichroism (CD) thermal denaturation data on vitellogenin promoter-binding protein (VBP), a member of the PAR subfamily of B-ZIP proteins that also includes thyroid embryonic factor, hepatocyte leukemia factor, and albumin site D-binding protein. VBP does not heterodimerize with B-ZIP domains from C/EBP alpha, JUND, or FOS. We describe a dominant negative protein, A-VBP, that contains the VBP leucine zipper and an acidic amphipathic protein sequence that replaces the basic region critical for DNA binding. The acidic extension forms a coiled coil structure with the VBP basic region in the VBP.A-VBP heterodimer. This new alpha-helical structure extends the leucine zipper N-terminally, stabilizing the complex by 2.0 kcal/mol. A-VBP abolishes DNA binding of VBP in an equimolar competition assay, but does not affect DNA binding even at 100-fold excess of CREB, C/EBP alpha, or FOS/JUND. Likewise, proteins containing the acidic extension appended to seven other leucine zippers do not inhibit VBP DNA binding. We show that conserved g <--> e' or i, i' +5 salt bridges are sufficient to confer specificity to VBP by mutating the C/EBPalpha leucine zipper to contain the g <--> e' salt bridges that characterize VBP. A-VBP heterodimerizes with this mutant C/EBP, preventing it from binding to DNA. These conserved g <--> e' electrostatic interactions define the specificity of the PAR subfamily of B-ZIP proteins and preclude interaction with other B-ZIP subfamilies.     

Computational approaches to identify leucine zippers.

The leucine zipper is a dimerization domain occurring mostly in regulatory and thus in many oncogenic proteins. The leucine repeat in the sequence has been traditionally used for identification, however with poor reliability. The coiled coil structure of a leucine zipper is required for dimerization and can be predicted with reasonable accuracy by existing algorithms. We exploit this fact for identification of leucine zippers from sequence alone. We present a program, 2ZIP, which combines a standard coiled coil prediction algorithm with an approximate search for the characteristic leucine repeat. No further information from homologues is required for prediction. This approach improves significantly over existing methods, especially in that the coiled coil prediction turns out to be highly informative and avoids large numbers of false positives. Many problems in predicting zippers or assessing prediction results stem from wrong sequence annotations in the database.     

A thermodynamic scale for leucine zipper stability and dimerization specificity: e and g interhelical interactions.

The leucine zipper is a dimeric coiled-coil protein structure composed of two amphipathic alpha-helices with the hydrophobic surfaces interacting to create the dimer interface. This structure has been found to mediate the dimerization of two abundant classes of DNA binding proteins: the bZIP and bHLH-Zip proteins. Several workers have reported that amino acids in the e and g positions of the coiled coil can modulate dimerization stability and specificity. Using the bZIP protein VBP as a host molecule, we report a thermodynamic scale (delta delta G) for 27 interhelical interactions in 35 proteins between amino acids in the g and the following e positions (g<==>e') of a leucine zipper coiled coil. We have examined the four commonly occurring amino acids in the e and g positions of bZIP proteins, lysine (K), arginine (R), glutamine (Q), glutamic acid (E), as well as the only other remaining charged amino acid aspartic acid (D), and finally alanine (A) as a reference amino acid. These results indicate that E<==>R is the most stable interhelical pair, being 0.35 kcal/mol more stable than E<==>K. A thermodynamic cycle analysis shows that the E<==>R pair is 1.33 kcal/mol more stable than A<==>A with -1.14 kcal/mol of coupling energy (delta delta Gint) coming from the interaction of E with R. The E<==>K coupling energy is only -0.14 kcal/mol. E interacts with more specificity than Q. The R<==>R pair is less stable than the K<==>K by 0.24 kcal/mol. R interacts with more specificity than K. Q forms more stable pairs with the basic amino acids K and R rather than with E. Changing amino acids in the e position to A creates bZIP proteins that form tetramers.     

Dual DNA recognition codes of a short peptide derived from the basic leucine zipper protein EmBP1

Sequence-specific DNA binding of short peptide dimers derived from a plant basic leucine zipper protein EmBP1 was studied. A homodimer of the EmBP1 basic region peptide recognized a palindromic DNA sequence, and a heterodimer of EmBP1 and GCN4 basic region peptides targets a non-palindromic DNA sequence when a beta-cyclodextrin/adamantane complex is utilized as a dimerization domain. A homodimer of the EmBP1 basic region peptide binds the native EmBP1 binding 5'-GCCACGTGGC-3' and the native GCN4 binding 5'-ATGACGTCAT-3' sequences with almost equal affinity in the alpha-helical conformation, indicating that the basic region of EmBP1 by itself has a dual recognition codes for the DNA sequences. The GCN4 basic region peptide binds 5'-ATGAC-3' in the alpha-helical conformation, but it neither shows affinity nor helix formation with 5'-GCCAC-3'. Because native EmBP1 forms 100 times more stable complex with 5'-GCCACGTGGC-3' over 5'-ATGACGTCAT-3', our results suggest that the sequence-selectivity of native EmBP1 is dictated by the structure of leucine zipper dimerization domain including the hinge region spanning between the basic region and the leucine zipper.     

The role of helix stabilizing residues in GCN4 basic region folding and DNA binding

Basic region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a coiled-coil leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The basic region is largely unfolded in the absence of DNA, but adopts a helical conformation upon DNA binding. Although a coil --> helix transition is entropically unfavorable, this conformational change positions the DNA-binding residues appropriately for sequence-specific interactions with DNA. The N-terminal residues of the GCN4 DNA-binding domain, DPAAL, make no DNA contacts and are not part of the conserved basic region, but are nonetheless important for DNA binding. Asp and Pro are often found at the N-termini of alpha-helices, and such N-capping motifs can stabilize alpha-helical structure. In the present study, we investigate whether these two residues serve to stabilize a helical conformation in the GCN4 basic region, lowering the energetic cost for DNA binding. Our results suggest that the presence of these residues contributes significantly to helical structure and to the DNA-binding ability of the basic region in the absence of the leucine zipper. Similar helix-capping motifs are found in approximately half of all bZip domains, and the implications of these findings for in vivo protein function are discussed.     

Dimerization of leucine zippers analyzed by random selection.

The leucine zipper is a coiled coil that mediates specific dimerization of bZIP DNA-binding domains. A hydrophobic spine involving the conserved leucines runs down the coiled-coil and is thought to stabilize the dimer. We used the method of random selection to further define the primary sequence requirements for homodimer formation and heterodimer formation with Fos. When positions on either side of the hydrophobic spine of GCN4 are diversified to include the corresponding residues of Jun, a large percentage of the resulting sequences form homodimers, and a large percentage form heterodimers with Fos. Basic residues were preferred, but not essential, at position e of zippers which heterodimerize with Fos. When random sequences containing 5 heptad repeat of leucines are subject to a selection for homodimer formation, a diverse set of sequences is isolated. Certain residues are preferred at each position in the heptad repeat, although no essential primary sequence determinants could be identified. No pair of residues not involving the conserved leucines could be identified which strongly promotes homodimerization. These results suggest that factors determining leucine zipper dimerization are complex, with numerous interactions contributing to the association.