Porte D, Oertel-Buchheit P, Granger-Schnarr M, Schnarr M
Institut de Biologie Moléculaire et Cellulaire du CNRS, UPR 9002, Strasbourg, France.
J Biol Chem. 1995 Sep 29;270(39):22721-30. doi: 10.1074/jbc.270.39.22721.
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.
Fos野生型亮氨酸拉链无法支持同源二聚化。这一发现通常被解释为Fos拉链的负净电荷导致两个单体之间的静电排斥。我们在此利用大肠杆菌中依赖LexA的体内分析表明，Fos拉链结合的额外反决定因素是Fos拉链界面内a位的残基。如果野生型Fos拉链与LexA阻遏物的DNA结合结构域（LexA-DBD）融合，与单独的LexA-DBD相比，未观察到额外的抑制作用，这与野生型Fos拉链促进同源二聚化的无能相一致。如果将疏水氨基酸（异亮氨酸、亮氨酸、缬氨酸、苯丙氨酸、甲硫氨酸）插入LexA-Fos拉链融合蛋白的五个a位，则恢复了显著的转录抑制，表明Fos拉链同源二聚化不仅受带负电荷残基的排斥限制，还受a位的非疏水性质限制。最有效的变体（在五个a位含有异亮氨酸或亮氨酸）与野生型Fos拉链融合蛋白相比，转录抑制增加了约80倍。在多个相同取代的情况下，总体改善与插入侧链的疏水性相关，即异亮氨酸>亮氨酸>缬氨酸>苯丙氨酸>甲硫氨酸。然而，至少对于缬氨酸、苯丙氨酸和甲硫氨酸，给定残基类型对结合效率的影响强烈取决于七肽，即取决于a残基的局部环境。这在Fos拉链的第二个七肽中尤为明显，其中缬氨酸的耐受性不如苯丙氨酸和甲硫氨酸。很可能a1残基调节了g1和e2位两个谷氨酸侧链之间的螺旋间排斥。大多数疏水Fos拉链变体与Jun亮氨酸拉链的异源结合也得到改善，使得同源二聚化额外自由能的大约一半被导入异源二聚体。一些候选物（包括Fos野生型拉链）偏离了这种相关性，表现出相当大的额外异源结合。
