FOS LEUCINE-ZIPPER VARIANTS WITH INCREASED ASSOCIATION CAPACITY

The Fos wild-type leucine zipper is unable to support homodimerization . This finding is generally explained by the negative net charge of th e Fos zipper leading to the electrostatic repulsion of two monomers, U sing a LexA-dependent in vivo assay in Escherichia coil, we show here that additional antideterminants for Fos zipper association are the re sidues 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 zippe r to promote homodimerization, If hydrophobic amino acids (ne, Leu, Va l, Phe, Met) are inserted into the five a positions of a LexA-Fos zipp er fusion protein, substantial transcriptional repression is recovered showing that Fos zipper homodimerization is not only limited by the r epulsion 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 tra nscriptional repression as compared with the wild-type Fos zipper fusi on protein, In the case of multiple identical substitutions, the overa ll improvement is correlated with the hydrophobicity of the inserted s ide 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 eff iciency 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 a, residue modulates the interhelical repulsion betwee n two glutamic acid side chains in positions g(1) and e(2). Most of th e hydrophobic Fos zipper variants are also improved in heteroassociati on with a Jun leucine zipper, such that roughly half of the additional free energy of homodimerization is imported into the heterodimer, A f ew candidates (including the Fos wild-type zipper) deviate from this c orrelation, showing considerable excess heteroassociation.