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INTRODUCTION |
Protein-protein complexes play crucial roles in many biological
processes such as gene expression, replication, DNA repair, signal
transduction, enzyme regulation, and immune response (1-9). The rules
determining specificity in protein-protein recognition are therefore of
fundamental biological importance. Among the structural motifs
mediating protein-protein recognition, assemblies of
-helices are
probably most widespread (10). Oligomerization of transcription
factors, e.g. is often accomplished by
-helical coiled
coils (11), and four-helix bundles form dimerization surfaces in many
proteins (12). The mechanistic details governing recognition
specificity and binding affinity of four-helix bundle mediated protein
oligomerization are only poorly understood. We investigated subunit
recognition in the four-helix bundle of Tet repressor
(TetR)1 dimers with the goal
of determining generally applicable rules for this process.
TetR sequence variants regulate tetracycline-induced expression of
seven naturally occurring tetracycline-resistance determinants (classes
A-E, G, and H; for a review, see Ref. 13). In the absence of
tetracycline, dimeric TetR binds to tet operator
(tetO) repressing transcription of the tet
promoters. Nanomolar concentrations of tetracycline lead to
dissociation of TetR from tetO to induce transcription. This
transcriptional switch is exceptionally sensitive to low inducer
concentrations, making it the system of choice for regulation of gene
expression in many higher organisms, including plants, transgenic mice,
and human cells (for two recent reviews, see Refs. 14 and 15).
Crystal structures of TetR(D) in complex with
[Mg-tetracycline]+ revealed a small N-terminal and a
large C-terminal domain (16, 17). The latter mediates tetracycline
binding and dimerization via a four-helix bundle formed by the helices
8 and
10 of each subunit. Disruption of the dimeric structure of
TetR by urea concomitantly leads to denaturation of the subunits
(18).
We have established that the sequence variants TetR(B) and TetR(D) do
not form heterodimers because amino acids located in the core of the
four-helix bundle in helix
10 of TetR(B) and TetR(D) lack structural
complementarity (19). Adjusting all amino acids in
10 yielded a
change in subunit recognition specificity but did not restore the full
dimerization efficiency of the wild type. We demonstrate here that this
requires only one additional exchange, E128R, located in helix
8 at
the edge of the four-helix bundle. Furthermore, we construct a
hyperstable TetR(B) variant by improving the interaction of Arg-128
with residues of the second monomer. Thus, interactions between
partially solvent-exposed, hydrophilic residues can profoundly
influence the specificity of subunit recognition in protein
hetero-oligomers and improve the stability of homo-oligomers.
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EXPERIMENTAL PROCEDURES |
Materials and General Methods--
Chemicals were obtained from
Merck (Darmstadt), Serva (Heidelberg), Sigma (München), or Roth
(Karlsruhe) and were of the highest purity available. Tetracycline was
purchased from Fluka (Buchs). Enzymes for DNA restriction and
modification were obtained from Boehringer Mannheim, Life Technologies,
Inc. (Eggenstein), New England Biolabs (Schwalbach), or Pharmacia
(Freiburg). Sequencing was carried out according to the protocol
provided by Perkin Elmer for cycle sequencing.
Bacterial Strains, Plasmids, and Phage--
All bacterial
strains are derived from Escherichia coli K12. Strain
DH5
(hsdR17
(rKmK+),recA1,endA1,gyrA96,thi,relA1,supE44,
80dlacZ
M15,
(lac ZYA-argF)U169) was used for general cloning procedures. Strain WH207
(lacX74,galK2,rpsL,recA13) served as host strain for
-galactosidase assays. The plasmids pWH1200 (20), pWH520
26-53 (21), pWH806, and pWH853 (22), pWH620
26-53, pWH853(B/D)51-208, pWH853(B/D)51-178,
pWH853(B/D)179-184, and pWH853(B/D)179-208 (19) as well as pWH1950
(23), which were used in the in vivo studies, for the
construction of tetR(B/D) mutants and overexpression have
been described.
Construction of tetR Variants--
The mutation E128R was
introduced into tetR(B) by polymerase chain reaction
according to a three-primer method (24). The conditions of the
polymerase chain reactions were as described (25). The products of the
second polymerase chain reaction were purified, digested with
MluI and BstEII, and cloned into likewise digested pWH853(B/D)179-208. The resulting pWH853 derivative was named
pWH853(B/D)128. MluI/NcoI or
MluI/BstEII fragments of
tetR(B/D)179-208 and tetR(B/D)179-184 were
cloned into likewise digested pWH853(B/D)128, yielding
pWH853(B/D)128,179-208 and pWH853(B/D)128,179-184, respectively. tetR(B/D)51-127,129-178 and
tetR(B/D)51-127,129-178,128A were constructed by introducing
the R128E or R128A mutations in tetR(B/D)51-178 by
polymerase chain reaction. pWH1950 derivatives were constructed for
tetR(B/D)128, tetR(B/D)179-184, and
tetR(B/D)128,179-184. The corresponding pWH853
derivatives were digested with XbaI and NcoI, and the tetR fragments were purified and
cloned into likewise digested pWH1950. DNA of positive candidates was
analyzed by digestion with restriction enzymes and sequencing of
tetR.
-Galactosidase Assays--
Repression and induction
efficiencies of the TetR variants as well as the negative
transdominance efficiencies of
tetR(B)
26-53 and
tetR(D)
26-53 were assayed in
E. coli WH207(
tet50). The phage
tet50 contains a tetA-lacZ transcriptional
fusion integrated as single copy into the WH207 genome (22). Bacteria
were grown in LB supplemented with the appropriate antibiotics.
Quantification of induction efficiencies was done with 0.2 µg/ml
tetracycline in overnight and log-phase cultures.
-Galactosidase
activities were determined as described by Miller (26). Three
independent cultures were assayed for each strain, and measurements
were repeated at least twice.
Purification of TetR Variants--
pWH1950 or pWH1950
derivatives were transformed into E. coli RB791. Cells were
grown in 3 liters of LB at 28 °C in shaking flasks. Tet repressors
were overexpressed by adding
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 1 mM at an A600 of
0.7-1.0. Cells were pelleted, resuspended in buffer A (50 mM NaCl, 2 mM dithiothreitol, and 20 mM Na3PO4, pH 6.8), and broken by
sonication, and TetR was purified by cation exchange chromatography and
gel filtration as described (23).
Determination of the in Vitro Stability of TetR
Variants--
Circular dichroism (CD) measurements were performed on a
Jasco J715 spectropolarimeter at protein concentrations of 5 µM in 0.5-cm cells. All measurements were carried out at
a temperature of 22 °C. Equilibrium denaturation was performed by
incubating protein samples overnight at the indicated urea
concentration. We used F-buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol) for all
spectroscopic measurements. Urea was obtained from ICN Biochemicals
(Eschwege), and urea solutions were prepared every day. Renaturation
was achieved by incubating the samples overnight at 8 M
urea and then diluting them 200-fold with F-buffer. Thermodynamic
calculations were done as described before (18).
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RESULTS |
Tet(B/D) Repressors--
Using chimeric TetR(B/D) repressors, we
demonstrated previously that residues in helix
10 are critical for
the different dimerization specificities of TetR(B) and TetR(D);
however, further changes are required to achieve WT affinity of subunit
binding (19). The monomer-monomer distances in the
TetR(D)/[(Mg-tetracycline)+]2 crystal
structure and a structural model of TetR(B) are different at residues
in
10 and at position 128 in
8 (19). Thus, we constructed five
new TetR(B/D) repressor dimers to analyze the influence of mutations at
position 128 on dimer formation. The sequences of these five dimeric
TetR(B/D) repressors and those from the previous study used here again
are depicted in Fig. 1. We introduced the
mutation E128R into TetR(B) and TetR(B/D)179-208. The resulting
repressors were named TetR(B/D)128 and TetR(B/D)128,179-208, respectively. TetR(B/D)51-127,129-178 and TetR(B/D)51-127,129-178,A128 were made to analyze whether the residue at position 128 influences dimerization specificity of TetR(B/D) repressors by attractive interactions with TetR(D) or repulsive interactions with TetR(B). These
TetR(B/D)51-178 derivatives contain either the Arg
Glu or the
Arg
Ala mutation at position 128. To evaluate if the interaction
of the TetR(D) residues at positions 128 and 179'-184' can be used to
construct a hyperstable protein, we also introduced the mutation E128R
in TetR(B/D)179-184 leading to TetR(B/D)128,179-184.

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Fig. 1.
Amino acid composition of Tet(B/D)
repressors. The TetR(D) amino acid sequence is given in the
one-letter code. Residues that build the dimerization surface in the
TetR(D)/([Mg-tc]+)2 crystal are marked by
triangles. Amino acids of TetR(B) that are identical to the
corresponding TetR(D) residue are indicated by dashes. The
lines below the sequence alignment indicate the parts of the
TetR(B/D) chimera, which are encoded by tetR(D).
Non-TetR(D) amino acids at position 128 are indicated by the one letter
code for the Tet(B/D) repressors. -Helices of the
TetR(D)/([Mg-tetracycline]+)2 crystal
structure are shown as rectangles above the
sequence alignment.
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In Vivo Repression and Induction Efficiencies--
The
tetR variants were transformed into E. coli
WH207
tet50 to test in vivo operator binding
activity and inducibility. pWH853 derivatives, which constitutively
produce low levels of TetR (22), were used as expression plasmids.
Repression of the tetA-lacZ transcriptional fusion and
inducibility by tetracycline were quantified at 37 °C. The results
are shown in Table I. The presence of WT TetR(B) results in repression of
-galactosidase activity to 2.4% of
that in the absence of TetR. All TetR(B/D) variants show slightly increased repression efficiencies. Repressor variants with different amino acids only at position 128 show similar repression efficiencies. All chimeric TetR(B/D) repressors were to the same extent inducible by
tetracycline as WT TetR. Thus, the E128R exchange does not impair
in vivo repression or inducibility of TetR.
The Mutation E128R Increases the Dimerization Efficiency of
TetR(B/D) Chimera with TetR(D) but Does Not Affect Dimerization with
TetR(B)--
Dimerization of TetR(B), TetR(B/D)128, TetR(B/D)179-208,
and TetR(B/D)128,179-208 with TetR(B) and TetR(D) was quantified to
check whether the E128R mutation affects protein recognition. Dimerization efficiencies were determined in assays of negative transdominance using TetR(B)
26-53 or TetR(D)
26-53 as
transdominance probes. Transdominance efficiencies of these mutants
over TetR(B/D) variants indicate the amount of dimer formation of the
TetR(B/D) variant with TetR(B) and TetR(D), respectively (19). The
results are also shown in Table I. Efficient dimerization of
TetR(B)
26-53 is only observed for TetR(B) and TetR(B/D)128. No
dimerization is detected between TetR(B)
26-53 and TetR(B/D)51-208,
TetR(B/D)179-208, or TetR(B/D)128,179-208. TetR(D)
26-53 dimerizes
only with TetR variants containing at least the TetR(D) amino acids
179-208. This confirms that the main determinants of the dimerization
specificities of TetR(B) and TetR(D) are located in helix
10 and
that the dimerization efficiency of TetR(B)
26-53 with TetR(B) is not
affected when E128R is present as a single exchange. The
dimerization efficiency of TetR(D)
26-53 is about 4-fold higher for
TetR(B/D)128,179-208 compared with TetR(B/D)179-208. In fact,
TetR(B/D)128,179-208 exhibits the same dimerization efficiency
with TetR(D)
26-53 than TetR(B/D)51-208, which contains the complete
TetR(D) protein core. These data demonstrate that only the E128R
exchange is needed to restore WT dimerization efficiency of
TetR(B/D)179-208 with TetR(D)
26-53.
Loss of Contact Analysis--
The increased dimerization
efficiency of Tet(B/D)128,179-208 with TetR(D) might be caused by an
attractive interaction of Arg-128 or a repulsive interaction of Glu-128
with the TetR(D) monomer. To distinguish between these two
possibilities, we investigated the effect of different residues at
position 128 on dimerization of TetR(B/D)51-178 with TetR(D).
TetR(B/D)51-178 was chosen because it forms dimers with TetR(B) and
TetR(D) (Ref. 19; Table I). We performed a loss of contact analysis by
mutating Arg-128 of TetR(B/D)51-178 to A128 in
TetR(B/D)51-127,129-178,A128 (Fig. 1). As a control, we also
constructed TetR(B/D)51-127,129-178, which contains the TetR(B) amino
acid Glu-128. Dimerization with TetR(D)
26-53 is neither detectable
for TetR(B/D)51-127,129-178,A128 nor for TetR(B/D)51-127,129-178. In
contrast, the dimerization efficiency of TetR(B)
26-53 with these
TetR variants is not affected (see Table I). Thus, the Arg-128 residue
increases the dimerization efficiencies of TetR(B/D) repressors with
TetR(D) by forming an attractive interaction with the other TetR(D) monomer.
Introduction of the TetR(D) Residues at Positions 128 and 179-184
Results in a Hyperstable TetR(B) Variant--
To test whether
the TetR(B) homodimer is stabilized by interaction of Arg-128 with
residues of the second monomer, we constructed TetR(B/D)128,179-184.
This variant contains Arg-128 and those TetR(D) residues which contact
Arg-128 across the dimerization surface. TetR(B/D)179-184 and
TetR(B/D)128,179-184 show identical dimerization efficiencies with
TetR(B)
26-53 and TetR(D)
26-53 (Table I). Thus, the E128R exchange
does not alter the dimerization specificity of TetR(B/D)179-184.
Urea-dependent Stability of TetR(B/D)
Variants--
The in vitro stabilities of TetR(B),
TetR(B/D)128, TetR(B/D)179-184, and TetR(B/D)128,179-184 were
determined by urea-induced unfolding. After unfolding in 8 M urea and refolding by a subsequent 200-fold dilution in
urea-free buffer, the CD spectrum of the respective TetR was identical
to that of the native protein (data not shown) and more than 92% of
the tetracycline-binding activity was recovered after the
unfolding/refolding cycle (Table II). We
conclude that denaturation under these conditions is reversible. The
urea-induced unfolding of the TetR(B/D) chimera as followed by the
change of the CD shows a monophasic, sigmoidal decrease (Fig.
2), and the midpoint of the unfolding
transition depends on the protein concentration (data not shown), as
expected for a bimolecular reaction. Thus, as for TetR(B) (18),
unfolding of the TetR(B/D) chimera is quantitatively described by a
two-state model, in which only folded dimers and unfolded monomers
exist at equilibrium in significant concentrations. Calculated
thermodynamic stabilities of the TetR(B/D) variants are also given in
Table II. The Gibbs free energy of unfolding of 75.2 ± 4 kJ
mol
1 determined for TetR(B) resembles the previously
published results (18). The stabilities of TetR(B/D)128 and
TetR(B/D)179-184 are identical to that of TetR(B). In contrast, a
stability increase of 12 kJ mol
1 is found for
TetR(B/D)128,179-184. These results demonstrate that the
urea-dependent unfolding stability of TetR(B) is only increased when positions 128 and 179'-184' are altered together, but
not when only either one of them is changed.

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Fig. 2.
Urea-induced denaturation curves obtained
from the change of the CD signal at 220 nm. The protein
concentration was 5 µM monomer, and the measurement was
carried out at 22 °C in 0.5-cm cells. The respective TetR variants
are: _ _, TetR(B) WT; , TetR(B/D)128; _, TetR(B/D)179-184; and
_ . . _, TetR(B/D)128,179-184.
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DISCUSSION |
Negative transdominance reflects the amount of dimer formed
in vivo by the TetR deletion mutants used as probes and the
TetR variant of interest (19). A comparison of the negative
transdominance efficiencies of TetR(D)
26-53 over
TetR(B/D)51-127,129-178, TetR(B/D)51-127,129-178,128A, and
TetR(B/D)51-178 as well as TetR(B/D)128,179-208 and
TetR(B/D)179-208 shows that mutations at position 128 influence the
dimerization potential of Tet(B/D) repressors with TetR(D).
Dimerization of TetR(B/D)51-178 with TetR(D) is abolished after
mutating Arg-128 to Glu or Ala, whereas that of TetR(B/D)179-208 is
increased after introducing the additional E128R exchange. The
dimerization efficiency of TetR(B/D)128,179-208 is almost identical to
that of TetR(B/D)51-208, which contains the complete TetR(D) protein
core. The single amino acid Arg-128 is essential for dimerization of
TetR(B/D)51-178 with TetR(D) and sufficient to restore WT dimerization
efficiency in TetR(B/D)179-208. Thus, no other TetR(D) residues than
Arg-128 and those in the segment 179-208 influence the dimerization
between Tet(B/D) repressors and TetR(D).
Dimerization of TetR(B) or TetR(B/D)179-184 with TetR(D) is not
affected by mutating Glu-128 to Arg-128. The mutation E128R alone is,
therefore, not sufficient for dimerization of TetR(B) with TetR(D).
This is in agreement with previous results, which identified the
amino acids 188, 192, 193, and 197 as the main determinants of
the TetR(B)/(D) dimerization specificity (19).
The mechanism of action by which Arg-128 influences dimerization with
TetR(D) is revealed by the mutations R128E and R128A in
TetR(B/D)51-178. Both mutations abolish dimerization with TetR(D). Because R128A is a loss of contact mutation, the increased dimerization efficiency of Tet(B/D) repressors containing Arg-128 with TetR(D) is
caused by a positive interaction and not a repulsive interaction of the
TetR(B) amino acid Glu-128 with the TetR(D) monomer.
In contrast to the dimerization with TetR(D), dimerization of TetR(B/D)
variants with TetR(B) is not affected by mutations at position 128. The
negative transdominance efficiencies of TetR(B)
26-53 over TetR
variants differing only in the residue at position 128
like TetR(B)
versus TetR(B/D)128, TetR(B/D)179-184 versus
TetR(B/D)128,179-184, and TetR(B/D)51-178 versus
TetR(B/D)51-127,129-178 or TetR(B/D)51-127,129-178,A128
are almost
identical. Thus, amino acids at position 128 are not important for
dimer formation with TetR(B), and no attractive interaction between the
amino acid at position 128 and the second monomer exists in the TetR(B) dimer.
The tetO-binding activities and inducibilities of the TetR
variants are only marginally affected by the mutations E128R, R128E, or
R128A. The mutants showing improved heterodimerization efficiency and
in vitro stability are functional. On the other hand, all TetR(B/D) mutants with a reduced dimerization efficiency show a loss of
function. This verifies that no major changes are introduced in the
structure of TetR. The properties of the mutations can, therefore, be
interpreted using the TetR(D) crystal structure. Arg-128 is located at
the N terminus of
8 in this structure and forms a hydrogen bond to
Gln-184' in
10, the two helices of the four-helix bundle
dimerization surface (Ref. 17; Figs. 3
and 4). Because positions 128 and 184 bear Glu and Pro in TetR(B), respectively, the hydrogen bond observed
in the TetR(D) crystal structure can neither be formed in the TetR(B)
dimer nor in heterodimers between TetR(D) and TetR(B/D) chimera
containing the TetR(D) amino acids of
10 and Glu-128. This explains
why mutations at positions 128 selectively affect dimerization of
Tet(B/D) repressors with TetR(D) but not with TetR(B).

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Fig. 3.
Stereo view of
TetR(D)-[(Mg-tetracycline)+]2
structure. Tetracycline is not shown. The helices are
depicted as ribbons. The two monomers are colored
yellow and gray, respectively, and the four-helix
bundle is highlighted by thicker lines. The side chain of
the solvent-exposed residue Arg-128 is shown in blue, and
the respective residues at 179'-184' in the other monomer are shown in
red.
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Fig. 4.
Proposed interaction of the TetR(D) residues
Arg-128 (blue) with Q184' (red).
The respective residues of TetR(B) are overlaid in orange
(Glu-128) and purple (Pro-184'). Helices belonging to different
monomers are shown in yellow and gray as in Fig.
3.
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