Abstract
The Fos wild-type leucine zipper is unable to support homodimerization. This finding is generally explained by the negative net charge of the Fos zipper leading to the electrostatic repulsion of two monomers. Using a LexA-dependent in vivo assay in Escherichia coli, we show here that additional antideterminants for Fos zipper association are the residues in position a within the Fos zipper interface. If the wild-type Fos zipper is fused to the DNA binding domain of the LexA repressor (LexA-DBD), no excess repression is observed as compared with the LexA-DBD alone, in agreement with the incapacity of the wild-type Fos zipper to promote homodimerization. If hydrophobic amino acids (Ile, Leu, Val, Phe, Met) are inserted into the five a positions of a LexA-Fos zipper fusion protein, substantial transcriptional repression is recovered showing that Fos zipper homodimerization is not only limited by the repulsion of negatively charged residues but also by the nonhydrophobic nature of the a positions. The most efficient variants (harboring Ile or Leu in the five a positions) show an about 80-fold increase in transcriptional repression as compared with the wild-type Fos zipper fusion protein. In the case of multiple identical substitutions, the overall improvement is correlated with the hydrophobicity of the inserted side chains, i.e. Ile Leu > Val > Phe > Met. However at least for Val, Phe, and Met the impact of a given residue type on the association efficiency depends strongly on the heptad, i.e. on the local environment of the a residue. This is particularly striking for the second heptad of the Fos zipper, where Val is less well tolerated than Phe and Met. Most likely the a1 residue modulates the interhelical repulsion between two glutamic acid side chains in positions g1 and e2. Most of the hydrophobic Fos zipper variants are also improved in heteroassociation with a Jun leucine zipper, such that roughly half of the additional free energy of homodimerization is imported into the heterodimer. A few candidates (including the Fos wild-type zipper) deviate from this correlation, showing considerable excess heteroassociation.
Highlights
Many transcription factors associate with their DNA binding site as either homodimers or heterodimers, making use of the so-called bZIP DNA binding motif
The Fos and Jun leucine zippers have been extensively studied in the past using both genetic and biophysical approaches
Homoassociation of Improved Hydrophobic Fos Zipper Variants—Here we describe Fos zipper variants with a modified hydrophobic interface, which are improved both in homoassociation and in heteroassociation with the Jun zipper as inferred from the recovery of repressor activity in the context of LexAFos zipper fusion proteins
Summary
Many transcription factors associate with their DNA binding site as either homodimers or heterodimers, making use of the so-called bZIP DNA binding motif. Granger-Schnarr et al [2] have shown recently that expression of a leucine zipper fused to the LexA DNA binding domain in transformed NIH3T3 fibroblasts leads to the recovery of an untransformed phenotype In this special case the target was a member of the AP-1 family of transcription factors. The preferential assembly of Jun and Fos as heterodimers has been shown to be mostly specified by the residues occupying the g and e positions [20] Among these residues two pairs of oppositely charged amino acids in positions g1 and e2 account for most of the additional free energy of heterodimerization [21]. In these positions the c-Fos protein, as well as the other members of the Fos family, harbors mostly polar residues, i.e. two threonine, two lysine, and one isoleucine residues
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