(Received for publication, April 26, 1995; and in revised form, July 17, 1995)
From the
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 a
residue
modulates the interhelical repulsion between two glutamic acid side
chains in positions g
and e
. 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.
Many transcription factors associate with their DNA binding site as either homodimers or heterodimers, making use of the so-called bZIP DNA binding motif. These proteins share the common feature of a basic region adjacent to a ``leucine zipper'' which is essential for dimerization (for a recent review, see (1) ). Most of them can form homodimers, and a subset forms heterodimers with other leucine zipper proteins. Leucine zippers are attractive targets to interfere with different cellular functions or dysfunctions. 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.
These factors are expressed in a variety of cell types, usually at low basal levels, and can be induced in response to a wide set of stimuli, including growth factors, oncogenes, cytokines and UV irradiation (for reviews, see (3) and (4) ). AP-1 consists of two groups of bZIP proteins, the Fos-related proteins (c-Fos, Fos B, Fra1, Fra2), and the Jun proteins (c-Jun, JunB, JunD). A Fos protein has to dimerize with a Jun protein in order to bind to DNA, whereas Jun forms both homodimers and heterodimers with other Jun and Fos proteins. Jun and Fos proteins interact further with several other transcription factors, giving rise to a cross-talk between different signal transduction pathways by the formation of alternative dimeric species. Jun and/or Fos interact via their leucine zippers with members of the ATF/CREB(5) , C/EBP(6) , Maf (7, 8) , and Rel families (9, 10) as well as MyoD(11) , FIP (12) and LRF-1(13) . Regions outside the leucine zipper seem to play a role in the interaction with the glucocorticoid receptor(14) . The composition of these mixed oligomers may dictate an altered DNA binding specificity (5, 15, 16, 17) or lead to the repression of transcription(11) .
The dimerization domains
of Jun and Fos proteins are -helical structures characterized by a
periodic repeat of leucine every 7th amino acid, i.e. every
two helical turns, which forms a parallel coiled-coil
structure(18, 19) . The seven amino acids of each
repeat are referred to by the letters a to g (a` to g` referring to the opposite helix). The d positions are occupied by the highly conserved leucine residues and
non-polar residues usually occur also at the a positions. Both a and d positions contribute to the formation of the
hydrophobic interface which accounts for most of the free energy of
dimerization. Additional stability can be provided by interhelical salt
bridges between oppositely charged residues at positions e and g.
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 gand e
account for most of the additional free
energy of heterodimerization(21) . The Jun protein forms
functional homodimers while the Fos protein does not. Beside
electrostatic repulsion, it has been suggested that Fos
homodimerization might also be hampered by a lack of hydrophobic
residues in the apositions(22) . In these positions the
c-Fos protein, as well as the other members of the Fos family, harbors
indeed mostly polar residues, i.e.two threonine, two lysine,
and one isoleucine residues.
To address the question if the
nonhydrophobic nature of these residues is indeed a major
antideterminant for Fos zipper dimerization, we have generated a
restricted random collection of Fos zipper mutants harboring Leu, Ile,
Val, Phe, or Met in the five a positions. The dimerization
capacities of these Fos zipper variants were assayed in a bacterial in vivo test using protein fusions between these zippers and
the LexA DNA binding domain(23, 24) , which is devoid
of intrinsic dimerization activity both free in solution (25, 26) and bound to operator DNA(27) . We
have used this assay previously to study Jun zipper variants mutated in
the e and g positions(28) . Similar approaches
made, respectively, use of the phage (29) and the phage
434 (30) repressor DNA binding domain.
Here we show that a
Fos leucine zipper can considerably improve the repressor activity of
the LexA DNA binding domain, if the a positions of the
coiled-coil are occupied by hydrophobic residues. The observed
improvement is correlated with the hydrophobicity of the inserted side
chain (Ile Leu > Val > Phe > Met) and a Fos zipper
variant can reach the efficiency of the Jun leucine zipper when all the a positions are occupied by leucine and isoleucine residues.
However the effect of a given residue depends strongly on the heptad
where it has been inserted. For example, Val is more penalizing than
Phe or Met if inserted in the a position of the second heptad of
the Fos zipper. Most likely the a residue in this heptad
modulates the interhelical repulsion between the two glutamic acid side
chains in positions g
and e
(21) .
Finally, we show that the most efficient homoassociating Fos zipper
variants are also improved in heteroassociation with the wild-type Jun
zipper. For most of the hydrophobic Fos zipper variants, one observes a
correlation between homoassociation and heteroassociation such that
roughly half of the additional free energy of homoassociation is
conserved upon interaction with the wild-type Jun zipper.
Plasmid pMS404 bearing the wild-type LexA-c-Fos zipper fusion was constructed by insertion of a BstEII restriction site into the XmnI site of pJWL70 using a short oligonucleotide. The C-terminal domain of LexA was deleted by BstEII/PstI digestion and the remaining N-terminal domain (residues 1-87) was fused to the Fos zipper. The DNA coding for this peptide (see Fig. 1A) was chemically synthesized and fused to the N-terminal part of lexA via the BstEII site, which contributes a valine in position 88.
Figure 1: Amino acid and nucleotide sequence of the Fos leucine zipper (A) and of the Jun leucine zipper (B) fused to the LexA DNA binding domain (amino acids 1-87). The nucleotide sequence has been chosen such that rare codons in E. coli are avoided and that several unique enzyme cleavage sites are present in the zipper coding sequences to allow for recombination of mutated subfragments.
Plasmid pDP107 bearing the LexA-c-Jun zipper fusion was obtained by insertion of a XhoI restriction site within the XmnI site of pJWL70. The C-terminal part of LexA was deleted by a XhoI/PstI digestion, and the remaining N-terminal domain was fused to the c-Jun zipper sequence shown in Fig. 1B. The XhoI site contributes a serine residue in position 88.
The single mutations in an
all-leucine context (Fig. 4) were obtained either by the
insertion of a small synthetic oligonucleotide cassette into the gene
of the randomly obtained mutant protein (Fos) or via
the recombination of several randomly obtained variants shown in Table 1(series pMS500) using the appropriate restriction enzymes.
Figure 4:
Positional effects of single mutations
(Ile, Val, Phe, or Met) in the context of an all-leucine hydrophobic
interface. The repression of the lacZ gene (strain JL1436,
10M IPTG) conferred by
LexA
-Fos zipper proteins varies according to the a
position, where the mutated residue has been incorporated. The penalty
index p
of each mutant protein relative to
the parental Fos
zipper (see ) is given in brackets behind the number of the experimentally determined
-galactosidase units (black bars). The gray bars are adjusted penalty values obtained upon optimization of the fits
of log(K
) versus log(K
) plots. For the LLMLL and LLLML
variants such an adjustment was not possible because the combinatorial
mutant set (Table 1) contains no methionine in positions a
and a
.
For the measurement of the heteroassociation capacity, a two-plasmid system was used allowing for a low expression of the proteins in order to minimize or to prevent the formation of homodimers. The expression of the lexA-c-junZIP gene was decreased by the introduction of a mutation within the ribosome binding site. This mutation (REG9) has been shown to reduce the level of LexA expression about 4-fold (35) and was introduced by an exchange of the ClaI/MluI restriction fragment of pDP107 (see above) for the ClaI/MluI restriction fragment of REG9 giving rise to plasmid pDP204. The lexA-c-junZIP gene was further transferred to a plasmid with lower copy number (pACYC184) which enables it to coexist with vectors that carry the colE1 origin of replication. The ClaI/DraI restriction fragment of pDP204 (750 base pairs) was fused to the pACYC184 vector after HincII/ClaI digestion (2550 base pairs), giving rise to plasmid pDP301.
The expression of the lexA-c-fosZIP wild-type and mutant genes was also decreased by exchanging the ClaI/MluI restriction fragment of pMS404 and of 15 candidates of the pMS500 series for the ClaI/MluI restriction fragment of REG9 giving rise to pMS604 and to the pMS600 set of plasmids for the wild-type and the mutant proteins, respectively.
The Fos proteins (385 amino acids) were purified by nickel affinity chromatography in the presence of guanidine hydrochloride(36) .
The ability of these domains to associate is tested as their
capacity to restore the repressor activity of the truncated LexA
protein (devoid of its own dimerization domain) in an in vivo test using appropriate E. coli indicator
strains(32) , one of which is schematized in Fig. 2A. The JL1436 host strain is deficient in the
chromosomal lexA gene and bears a lacZ gene driven by
the LexA-dependent sulA promoter, whereas in the JL806 strain,
the lacZ gene is under the control of the stronger (also
LexA-regulated) recA promoter. The repressor activity of the
fusion protein is determined as the residual -galactosidase
activity that can be measured at different levels of expression of the
proteins obtained upon varying the IPTG concentration. Characteristic
repression curves are shown in Fig. 2B. In the absence
of repression (expression vector alone), we measured about 4200
-galactosidase units regardless of the IPTG concentration. When
the truncated form of LexA (DBD alone) was expressed, a significant
repression of the lacZ gene was observed only upon
overproduction of the protein, reaching about 2300
-galactosidase
units at 10
M IPTG. With the entire LexA
protein, the lacZ gene is nearly completely turned off even in
the absence of IPTG. In the absence of IPTG the c-Jun zipper is able to
restore the DNA binding capacity of the truncated LexA protein at least
partially, whereas the c-Fos zipper does not. Upon overproduction of
the fusion protein, a level of repression identical to that obtained
with LexA was reached with the LexA-c-JunZIP protein, whereas with its
c-FosZIP counterpart the repression was the same as with the LexA DNA
binding domain alone. These results are in agreement with previous
results showing that (unlike the Jun zipper) the Fos zipper is unable
to promote homodimerization.
Figure 2:
A, schematic representation of the JL1436 E. coli indicator strain. This strain harbors a single copy of
the lacZ gene under the control of the promoter/operator
region of the sulA gene which is under the control of the LexA
repressor. The strain is deficient in the chromosomal lexA gene and transcription of the lacZ gene will be repressed
only, if the LexA deficiency is complemented by a plamid producing a
functional LexA-leucine zipper fusion protein. The
production of these fusion proteins is further regulated by the E.
colilac repressor (produced from an F` factor) and is
thus IPTG inducible. Strain JL806 is essentially identical except that
the lacZ gene is under the control of the recA promoter/operator region. B, representative repression
curves as a function of IPTG concentration, using strain JL1436. The
unrepressed state (⊡, pBR322) gives rise to about 4200
-galactosidase units. Transformation with a plasmid coding either
for the LexA DNA binding domain alone (
) or for
LexA
-c-FosZip (
) gives rise to the same
degree of repression at elevated IPTG concentration. Transformation
with a plasmid coding for a LexA
-c-Fos
(
) fusion protein (i.e. all a positions of the Fos
zipper are replaced by leucine residues) gives rise to a degree of
repression similar to a LexA
-c-JunZip protein
(
). The intact wild-type LexA protein (
) remains the
strongest repressor at least at low IPTG
concentration.
Figure 3:
Correlation between the repression of the lacZ gene in the two indicator strains JL1436 (sulA promoter) and JL806 (recA promoter) at 10M IPTG: hydrophobic LexA
-Fos zipper
variants (
) harboring only Leu, Ile, Val, Phe, or Met in
position a (see Table 1), LexA
-c-FosZip
(
), LexA DNA binding domain (
).
Given that for all the fusion proteins the LexA-DBD is unchanged, the increased repressor activity is likely to reflect the efficiency with which the different leucine zippers are able to associate, provided that the proteins are produced in similar amounts. Therefore for each mutant protein, the relative in vivo amount was measured (at least in duplicate) by an immunoblot analysis using antibodies against the LexA part of the chimeric proteins as described under ``Materials and Methods.'' The intracellular steady-state level of all the mutant proteins were found similar or identical to the parental LexA-c-FosZIP protein.
Table 1shows
the collection of Fos zipper variants classified according to the
amount of -galactosidase units measured in strain JL1436. The
smaller the number of units, the tighter the transcription of the
indicator gene is repressed by the LexA-Fos zipper fusion proteins.
Under these conditions the LexA-DBD alone and the LexA-Fos wild-type
zipper fusion (TTKIK in Table 1) give rise to about 2300 units.
The most strongly improved variants contain mostly Ile or Leu side
chains in the five a positions, giving rise to only 28 units in
the case of five Ile residues, i.e. an 82-fold improvement in
repressor efficiency as compared with the Fos wild-type zipper fusion
protein.
The different hydrophobic side chains are not equivalent in
promoting Fos zipper association. Phenylalanine and methionine
residues, especially, are mostly found within weakly improved Fos
zipper variants, suggesting that these amino acids are not as efficient
as leucine, isoleucine, or valine side chains (see Table 1). In
order to establish this hierarchy more precisely Fos zipper variants
were created bearing the same hydrophobic amino acid within the five a positions (Table 1, bottom right). As anticipated from
the random collection we find the following hierarchy: Ile Leu
> Val > Phe > Met. This order may be understood in terms of
the hydrophobicity of these side chains (see Discussion).
As shown in Fig. 4, Ile is at least as efficient as Leu, since in none of
the five a positions it influences the dimerization capacity of
the Fos host zipper. This is not the case for the other
three amino acids, which all, to various degrees, diminish the
repressor activity of the LexA-Fos
protein.
As
suspected from the random combinatorial variants, we observe pronounced
position-dependent effects. The a position of the first heptad
is less sensitive to mutations than the four others, since only minor
changes are observed even when ais occupied by the
relatively unfavorable residues Phe and Met. This may be understood by
the fact that a
is not packed between two leucine layers
as the other aresidues (see Fig. 8B).
Figure 8:
Correlation between the square root of the
relative dissociation constant for homoassociation (deduced from the
-galactosidase units in Table 1) and the relative
dissociation constant for heteroassociation (deduced from the
-galactosidase units in Fig. 7).
represents the
wild-type Fos zipper,
the outsiders VMIIM and VVIFV, and
the other variants from Fig. 7.
Figure 7:
Repression of the lacZ gene
(JL1436, no IPTG) due to heteroassociation of
LexA-Fos zipper variants with the
LexA
-Jun zipper protein. The expression of the
fusion proteins was chosen such that a single fusion protein (gray
bars) gives rise to essentially no repression, whereas
coexpression of a Fos zipper variant with the Jun wild-type zipper
fusion protein (black bars) recovers repression. The
unrepressed state (4200 units ± 10%) is indicated by two
dashed lines.
In
position athe hierarchy (Ile
Leu >Val >Phe
>Met) mentioned above is inverted, i.e.in this position
Val is less favorable than Phe and Met. This atypical behavior is most
likely linked to the two glutamic acid residues in positions g
and e
`, which tend to pack over the a
side chain as discussed below. In a
and a
, Val is well accepted, whereas in a
it
is again unfavorable, albeit less than in a
. Phe and Met
are always unfavorable as compared with the parental Fos
zipper, albeit with some modulations. Phe is particularly
unfavorable in a
and Met in a
. Hu et
al.(29) also observed positional effects in aand dpositions in a study of the GCN4 leucine zipper.
No crystal structure is available for a leucine zipper harboring Leu in both a and d. Zhu et al.(40) have shown by size exclusion chromatography for a coiled-coil with Leu residues at positions a and d that in the reduced form these peptides adopt a dimeric (two-stranded) coiled-coil structure. Harbury et al.(38) showed (also by size exclusion chromatography) that a GCN4 leucine zipper variant harboring Leu at positions a and d apparently forms trimers in the reduced state, but dimers and higher oligomers in the oxidized (disulfide-bonded) state. This behavior is not easily understood in comparison with an isoleucine zipper (Ile in a and d), which forms also trimers in the reduced state, but only hexamers in the oxidized state(38) .
To address the question if a Fos
leucine zipper variant harboring Leu at positions a and d would have a strong tendency to form higher order species, we have
constructed and purified Fos protein variants harboring mostly or
exclusively leucine or isoleucine in position a (for details see
``Materials and Methods''). Fig. 5shows that contrary
to the Fos protein harboring a wild-type leucine zipper (lane
4), the Fos variants Fos (lane 1),
Fos
(lane 2), and Fos
(lane
3), harboring a hydrophobic interface, are able to interact with a
21-base pairs AP-1 binding site (TGACTCA).
Figure 5:
Electrophoretic mobility shift assay
showing that full-length Fos protein variants harboring a hydrophobic
leucine zipper interface are able to interact with a 21-mbase pair DNA
duplex harboring an AP-1 binding site (TGACTCA): Fos (lane 1), Fos
(lane 2), Fos
(lane 3); whereas a Fos protein harboring the wild-type
leucine zipper (lane 4) does not bind to this oligonucleotide.
The protein concentration was 1.15
10
M and the DNA concentration about 5
10
M. For the assay conditions, see ``Materials and
Methods.''
Relevant for the question
we ask here is the finding that the gel mobility of the two complexes
with a leucine interface (Fos and Fos
) is the
same as that with an isoleucine interface (Fos
). This
behavior strongly suggests that the dominant species for both proteins
should be a protein dimer, since Ile in position a (with Leu in
position d) is known to favor the formation of leucine zipper
dimers as shown by equilibrium ultracentrifugation(38) . Gel
retardation is in fact very sensitive to the oligomeric state of a
protein bound to a small DNA duplex. For example the migration of a
dimeric variant of lac repressor (a protein of 360 amino
acids, i.e. of similar size as the Fos protein) is different
from that of the tetrameric repressor in a complex with a 40-base pair
operator fragment (41) .
For the following reason the data in Table 1also argue in favor of the dimer as the dominant oligomerization state of the LexA-c-FosZip variants in vivo; if the repressor effect of the multiple combinatorial mutations in Table 1could be understood in terms of the sum of the corresponding single mutations (placed in a Leu hydrophobic interface, see Fig. 4), we may argue that multiple oligomerization states should not play a major role in the establishment of the repressor efficiency (Table 1), since (contrary to the single mutations) the combinatorial mutations are not particularly leucine-rich, but contain also Ile (a dimer-driving residue in position a), as well as Val, Met, and Phe.
To test this hypothesis we have proceeded
in the following four steps. 1) The -galactosidase units of the 20
different single mutations were normalized to the all-leucine zipper
(Fos
) to obtain a relative dissociation constant
K
= K
/K
for each single
mutation in this context. In the case of the single mutations these
relative dissociation constants may be considered as a penalty index p
, associated with the mutation of a given
leucine to isoleucine, valine, phenylalanine, or methionine.
where is the fractional occupancy of the sulA operator by one of the LexA-mutant Fos zipper hybrid
proteins,
the fractional occupancy by the
LexA-c-Fos
protein, Z
the
number of
-galactosidase units of a single mutant hybrid protein
(see Fig. 4), Z the corresponding value for the
Fos
hybrid protein (40 units), and Z
the number of
-galactosidase units of the unrepressed state
(4183 units).
2) For each of the multiple mutations in Table 1we calculated a theoretical relative dissociation constant
with respect to the Fos hybrid
protein,
as the product of the corresponding K values, since if the effects of the single mutations are additive
in terms of loss in free energy, the corresponding relative equilibrium
constants have to be multiplied.
3) For each of the multiple
mutations in Table 1we calculated further an experimental
relative dissociation K constant with
respect to the Fos
hybrid protein according to the
following equation.
4) The theoretical relative dissociation constants were plotted
as a function of the experimental relative dissociation constants on a
log-log scale to guarantee an even distribution of the data points
along the 2 orders of magnitude covered by the experimental relative
dissociation constants. These log (K) versus log (K
) plots were finally
submitted to a linear least squares fitting procedure.
Fig. 6shows such a log-log plot of the theoretical Kversus the experimental K
value. A linear least squares fit of these
data gives the following straight line: logK
= 1.07 logK
- 0.15, with R
= 0.83.
Figure 6:
Correlation between the theoretical
relative dissociation constant (see ) and the experimental
relative dissociation constant K calculated according to . For the calculation of
K
the penalty values of the five
isoleucine variants were set to unity, since the deviation from the
parental Fos
mutant zipper are within experimental
error (see Fig. 4).
The slope (1.07) is close to the theoretically expected value of 1, and the intersection of the y axis is close to zero. This behavior shows that the experimental data scatter almost evenly around the expected correlation line. On average the multiple leucine zipper mutations may thus be explained by the additive effect of the individual mutations in the context of an all-leucine hydrophobic interface. This behavior suggests indeed that the repressor efficiency of the Lex A-Fos zipper fusion proteins in vivo is dominated by a single oligomerization state. This dominant state is most likely a Fos zipper dimer, since those leucine zippers which are rich in Ile in position a have been shown to form exclusively dimers(38) .
The correlation
between K and K
can be further improved by adjusting the
K
values. Adjustment was achieved by a
stepwise variation of each K
value and
subsequent recalculation of the correlation between
K
and K
. Using
an adjusted K
data set (see gray
bars in Fig. 4), the correlation (not shown) can be
increased to R
= 0.90. Major improvement is
achieved upon increasing the K
values of
MLLLL and LFLLL and diminishing that of LLLFL. Most of the other
K
values (especially those of the valine
single mutations) are close to their optimal value in terms of their
fitting efficiency.
Fig. 7shows the results obtained with a representative subset of the combinatorial Fos zipper variants of Table 1. Under these conditions the LexA-c-JunZip protein is not able to repress the lacZ gene in JL1436 and also most of the LexA-c-FosZip variants fall into the range of the unrepressed state, i.e. 4183 units ± 10%. Only two c-FosZip variants (LLLLL and ILLLL) show a slight residual repression due to homodimerization.
The coexpression of LexA-c-JunZip and
LexA-c-FosZip leads to the recovery of transcriptional repression. The
two wild-type zipper sequences together produce an about 3-fold
repression, whereas coexpression of LexA-c-JunZip with the
Fos variant leads to a 10-fold repression under these
conditions. Interestingly the four Fos zipper variants in Fig. 7being most improved in heteroassociation with the Jun
zipper (i.e. LLLLL, IILII, LIVVL, and ILLLL) are also the most
efficient homoassociating species of this set of Fos zipper variants.
Fig. 8shows that 13 of the 15 Fos
zipper variants are indeed reasonably close to a straight line in such
a plot (i.e. (K)
= 0.67 K
+ 0.29, R
= 0.83). In principle this straight line
should go through zero and have a slope of 1. A minor adjustment of the
exponent (i.e. 0.56 instead of 0.5 in the case of a square
root) leads indeed to a fit which fulfills these two requirements
better (i.e. (K
)
= 0.85 K
+ 0.01, R
= 0.83).
Two Fos zipper variants
(especially VVIFV, but also VMIIM) are fairly far apart from this
hetero- versus homoassociation correlation. Both variants
heteroassociate more efficiently than they should do in view of their
homoassociation capacity. The same holds true even more strikingly for
the Fos wild-type zipper (see Fig. 8, full circle),
which has the by far weakest homoassociation capacity of all the
variants shown in Fig. 7, but an intermediate heteroassociation
capacity as compared with the mutant Fos zippers. These data suggest
that one or several of the wild-type Fos zipper amino acids in position a (Thr, Thr, Lys, Ile, Lys) should be favorable for the
formation of heterodimers with the Jun zipper. The two lysine residues
(Lys) in positions aand a
are likely
candidates, since the crystal structure of the Fos/Jun DNA complex (19) shows that these residues form hydrogen bonds with two
glutamine side chains in positions g
and g
of the Jun zipper. The conservation of the five aresidues
Thr, Thr, Lys, Ile, Lys in c-Fos, Fos B, Fra1, and Fra2 (1) would be consistent with a possible role of aresidues in Fos/Jun zipper assembly.
The Fos and Jun leucine zippers have been extensively studied in the past using both genetic and biophysical approaches. Most of the mutagenesis work has been focused on loss-of-function mutations, which established the importance of the conserved leucine side chains for dimerization and DNA binding using either single or multiple hydrophobic (valine, phenylalanine) or helix breaking substituents(22, 43, 44, 45) .
A gain-of-function mutation has been described in the case of the Fos zipper (46) where the replacement of Glu-168 (which is the g residue of the first heptad) by lysine enables a truncated Fos protein to interact with DNA as a homodimer. For technical reasons the interaction of this variant with Jun could not be evaluated. It seems, however, unlikely that Fos E168K would be also improved in Jun binding, since in the Fos-Jun heterodimer this mutation would juxtapose two lysine residues.
The degree of improvement of these variants
in homoassociation is, however, not uniform and depends both on the
nature of the hydrophobic amino acid substituents and on the position
within the Fos zipper. If one compares Fos zipper variants harboring
only Leu or Ile or Val or Phe or Met in the five a positions,
then the improvement may be correlated with the hydrophobicity of the
side chains (see Fig. 9A). No correlation exists
between homoassociation improvement and -helix propensity of these
residues, since all relevant helicity scales (compiled in (48) ) attribute a higher
-helix propensity to Met (the
least efficient homoassociation-inducing residue in our experiments) as
compared with Ile (the most efficient residue in our experiments).
Figure 9: A, correlation between the homoassociation capacity of Fos zipper variants harboring only either leucine (L), isoleucine (I), valine (V), phenylalanine (F), or methionine (M) in the five a positions with the hydrophobicity of these side chains as inferred from the free energy of transfer from cyclohexane to water(45) . B, representation of a Fos homodimer (with variable a positions) as two parallel helical cylinders as adapted from Hu et al.(29) . Coupling between g residues in one helix with e` residues in the opposite helix is indicated by white bars.
The hydrophobicity scale used for the plot in Fig. 9A is from Radzicka and Wolfenden (49) and corresponds to the
free energies of transfer of the amino acid side chains from
cyclohexane to water. On this scale Leu, Ile, Val, Phe, and Met are the
five most hydrophobic amino acid side chains. The compilation of 61
leucine zipper-containing transcription factors (1) reveals
that 24% of the a positions are occupied by Val, 21% by Asn, 11%
by Leu, and 10% by Ile. The residues being on average most efficient in
promoting Fos zipper homodimerization (Ile Leu > Val) are thus
also beside the most common a residues in this leucine zipper
compilation.
The correlation between hydrophobicity and repressor efficiency shown in Fig. 9A corresponds to five simultaneous mutations in position a. Since the five heptades of the Fos zipper are not identical these data correspond to an average over different local environments.
What are
the possible reasons for this inversion from the general Val > Phe
> Met hierarchy to Phe > Met > Val in heptade 2? The most
likely explanation is that in heptade 2 the a residue modulates
the interhelical repulsion of the two glutamic acid side chains in
positions gand e
`(where the
primed residue corresponds to the opposite helix, see Fig. 9B), since in the case of the Fos zipper mutants,
the cavity into which an a
residue has to fit is the
same for a
, a
, a
, and a
. In a parallel coiled-coil the packing of the side
chains in the dimer interface is such that an aresidue is
surrounded by four amino acid side chains of the opposite
helix(50, 51) . In the Fos zipper an a
residue is always surrounded by a glutamic acid side chain (in
position g
`), two leucines (in d
`and d
`), and the
opposite a`residue, which is conserved in the case of a
homodimer (see Fig. 9B).
Whereas the cavity is the
same (including the g residues), the e residues are
variable from one heptade to the other. Host-guest studies have shown
that electrostatic repulsion between glutamic acid residues in
positions gand e
` is particularly
destabilizing for the host leucine zipper(21) . The atypically
behaving a
residue lies just between these residues and
may conceivably modulate the negative interaction between them. Valine
might bring the two glutamic acid side chains close together and thus
increase electrostatic repulsion. This hypothesis might also explain a
puzzling result of Krylov et al.(52) who showed that
the g`
e`coupling energy for glutamine pairs is
surprisingly favorable if packed over valine residues in position a. Physical model building based on the co-ordinates of the GCN4
leucine zipper (50) showed us that two glutamine residues (or
two protonated glutamic acid side chains) in gand e`of
the next heptade might be brought sufficiently close together to form
potentially two interhelical hydrogen bonds. Independently of the
precise molecular mechanism, our results strongly suggest that aresidues, especially valine, may modulate interhelical g`
e`coupling.
It is worthwhile to notice that a mutation of
valine in position aof the Jun leucine zipper to
phenylalanine gave rise to a more active Jun protein(53) ,
whereas the substitution of other aresidues (Asn in a
, Ala in a
, and Val in a
)
with phenylalanine was penalizing. In the Jun leucine zipper, a
lies also between equally charged residues (two
lysines in g
and e
`), which may also give
rise to electrostatic repulsion, albeit with a smaller thermodynamic
cost as in the case of two glutamic acids(21, 52) .
For these Fos zipper variants, one may conclude that the general stability rather than the specificity of Fos/Jun zipper assembly is affected. However we would not expect that these Fos zipper variants would be able to dimerize efficiently with any leucine zipper, since the major specificity determinants for Fos/Jun zipper assembly in positions e and g are conserved(20, 21) .
However there are a few
interesting exceptions from this correlation. Especially the wild-type
Fos zipper does not fit to this correlation. It has an intermediate
heteroassociation activity in this assay, but confers no
homoassociation at all, suggesting that the Fos wild-type a positions (TTKIK) are extremely unfavorable for homoassociation,
but acceptable or favorable for an interaction with the Jun leucine
zipper. Another exception from the correlation in Fig. 8is the
VVIFV variant. This hydrophobic Fos zipper variant is also better in
heteroassociation than expected from the homoassociation data. A
possible reason for this behavior is that the underlined valine
residues in positions aand a
are
destabilizing for a Fos zipper in the context of the equally charged
glutamic acid residues in positions g
, e
,
and g
Several canonical Fos zipper a variants (i.e. those for which homo- and heteroassociation are correlated) and the ``excess heteroassociation'' variants are currently under study for their transformation suppressor activity in ras-transformed fibroblasts.