Tài liệu Báo cáo khoa học: Tet repressor mutants with altered effector binding and allostery docx

Tet repressor mutants with altered effector binding and
allostery
Eva-Maria Henßler, Ralph Bertram, Stefanie Wisshak and Wolfgang Hillen
Lehrstuhl fu
¨
r Mikrobiologie, Institut fu
¨
r Biologie, Friedrich-Alexander Universita
¨
t Erlangen-Nu
¨
rnberg, Erlangen, Germany
Tetracycline (tc) resistance in Gram-negative bacteria
is often regulated by the Tet Repressor (TetR), a tc-
responsive allosterical DNA-binding protein. Due to
three very advantageous properties, namely the highly
specific binding of TetR to tet operator (tetO), the sen-
sitive induction by small amounts of tc, and the ability
of this drug to penetrate into most cells, the TetR
based regulation systems are widely used for condi-
tional gene expression [1]. Many biochemical studies
and crystal structures of TetR in all complexed forms
[2–4] have led to a detailed understanding of the regu-
latory mechanism.
TetR is an all a-helical, dimeric protein in which
tetO recognition is accomplished by a helix-turn-helix
motif consisting of helices a2 and a3 at the N-termi-
nus. The core domain (a5toa10) contains the tc bind-
ing pocket and the dimerization motif. Both domains
are connected by helix a4, and their interface is formed
by residues of helices a1, a4, and a6 (Fig. 1A). As

the [tc-Mg]
+
binding site is 33 A
˚
away from the tetO
binding site, the structural changes associated with
induction of TetR must be transfered through the pro-
tein. They are initiated at the residues 100–103 which
are part of helix a6 in the DNA-binding conformation
and assume a type II b-turn to contact [tc-Mg]
+
in the
induced state. The transmission of structural changes
to the DNA binding domains occurs via helices a4
and moves them by about 5° in a pendulum-like
motion so that the recognition helices no longer fit
into successive major grooves of DNA (Fig. 1B).
Extensive mutagenesis employing powerful selection
and screening systems have led to many TetR variants
with new activities. Among them was a TetR variant
with changed inducer specificity [5]. Instead of tc or the
more powerful inducer anhydrotetracycline (atc) the
TetR H64K S135L S138I triple mutant (TetR
i2
) recogni-
zes 4-de-dimethylamino-anhydrotetracycline (4-ddma-atc,
see Fig. 1C for chemical structures) [5,6], an analog
lacking the dimethylamino grouping at position 4 and
showing no antibiotic activity. In another effort TetR
Keywords

allostery; effector specificity; reverse TetR;
tetracycline derivatives; Tet repressor
Correspondence
W. Hillen, Lehrstuhl fu
¨
r Mikrobiologie,
Institut fu
¨
r Biologie, Friedrich-Alexander
Universita
¨
t Erlangen-Nu
¨
rnberg,
Staudtstraße 5, 91058 Erlangen, Germany
Fax: +49 9131 ⁄ 85 28082
Tel: +49 9131 ⁄ 85 28081
E-mail:
(Received 12 May 2005, revised 12 July
2005, accepted 15 July 2005)
doi:10.1111/j.1742-4658.2005.04868.x
To learn about the correlation between allostery and ligand binding of the
Tet repressor (TetR) we analyzed the effect of mutations in the DNA read-
ing head–core interface on the effector specific TetR
i2
variant. The same
mutations in these subdomains can lead to completely different activities,
e.g. the V99G exchange in the wild-type leads to corepression by 4-ddma-
atc without altering DNA binding. However, in TetR
i2

it leads to 4-ddma-
atc dependent repression in combination with reduced DNA binding in
the absence of effector. The thermodynamic analysis of effector binding
revealed decreased affinities and positive cooperativity. Thus, mutations in
this interface can influence DNA binding as well as effector binding, albeit
both ligand binding sites are not in direct contact to these altered residues.
This finding represents a novel communication mode of TetR. Thus, allo-
stery may not only operate by the structural change proposed on the basis
of the crystal structures.
Abbreviations
Atc, anhydrotetracycline; 4-ddma-atc, 4-de-dimethylamino-anhydrotetracycline; tc, tetracycline; TetR, tetracycline repressor; b-Gal,
b-Galactosidase.
FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS 4487
was converted to reverse TetR (revTetR) by single or
multiple mutations affecting the allostery of the protein.
RevTetR variants bind tetO only in the presence of tc,
thus turning the inducer into a corepressor [7,8]. The
underlying mutations occur in residues located in the
interface of the core and DNA binding domains which
do not move upon induction (Fig. 1A shows their loca-
tion in the TetR structure). It has therefore been
assumed that they may cause a repositioning of the
DNA reading head with respect to the core of TetR, so
that the same allosterical change as in wild-type could
result in the opposite activity [8].
The combination of mutations resulting in 4-ddma-
atc specificity with those yielding revTetR in the same
polypeptide did not yield efficient mutants with com-
bined activities [9]. Thus, these properties of TetR
must be interrelated. We use here the TetR

i2
variant
exhibiting 4-ddma-atc specificity to combine it with
degenerations of residues giving rise to revTetR
mutants and screen for the combined phenotype. The
results lead to insights about the allostery of TetR.
Results
Randomization of residues in helices a1, a4,
or a6 in TetR
i2
As the most efficient and mechanistically most interest-
ing mutations leading to revTetR occurred in helices
a1, a4, and a6 [8], we decided to combine randomiza-
tions in these helices with TetR
i2
(containing the muta-
tions H64K, S135L and S138I). We screened the
resulting candidates for TetR variants with 4-ddma-atc
specific corepression in Escherichia coli WH207 ⁄ ktet50
[10]. The specificity is scored against atc, the most
efficient effector of TetR known so far. The DNA
fragments containing the randomized codons 14–25
(C-terminal part of helix a1 and the following loop)
and 93–102 (helix a6 and the b-turns N-terminal and
C-terminal of a6) as described previously were intro-
duced into pWH1925-tetR
i2
. The randomized codons
50–63 in helix a4 generated by PCR mutagenesis using
a ‘doped’ oligonucleotide also encoding the H64K

mutation were as well inserted in pWH1925-tetR
i2
.
The three mutant pools were screened for repression in
the presence of 0.4 lm 4-ddma-atc and rescreened for
induction with 0.4 lm atc and without inducer on
MacConkey agar plates. We screened 17 600 colonies
with mutations in helix a1, 29 840 with mutations in
helix a4 and 3900 out of the helix a6 pool and
obtained a total of 15 candidates with the desired
properties. These were confirmed by in vivo repression
and induction determined in broth cultures of E. coli
B
A
C
Fig. 1. Structural depiction of the allostery in TetR. (A) Crystal struc-
ture of the TetR-[tc-Mg]
+
2
complex. One monomer is shown as
dark blue ribbon, the second monomer in light blue. Tc is drawn as
a yellow stick model. The mutated parts of helices a1, a4anda6
(see arrows) forming the interface between the DNA-binding head
and the protein core are highlighted in red in one subunit. (B) Over-
lay of the induced (dark blue) and tetO bound (grey) partial struc-
tures of one TetR subunit. Tetracycline is depicted as a yellow stick
model. Leu17 (in helix a1), Val99 and Thr103 (in helix a6) and
Leu52, Leu56 and His64 in helix a4 are indicated in the induced
structure by red side chains and S135 in helix a8 by the green side
chain. All amino acids are designated in the three letter code. The

C-terminus of helix a4 is connected to the [tc-Mg]
+
binding pocket
by interaction of His64 with tc. Leu52 and Leu56 form the hydro-
phobic region contacting Val99. Thr103 is located in the C-terminal
helical turn of a6 which is transformed into a type II b-turn upon
induction [20]. (C) Chemical structures of anhydrotetracycline (atc)
and 4-de-dimethylamino-anhydrotetracycline (4-ddma-atc).
Altered TetR effector binding and allostery E M. Henßler et al.
4488 FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS
WH207 ⁄ ktet50 containing a chromosomal tetA-lacZ
fusion as summarized in Table 1.
Three candidates with mutations in helix a1 exhibit
4-ddma-atc dependent repression, but show different
specificities as scored by the lack of atc-mediated repres-
sion. TetR
i2
-E15A L17V shows repression to 12.5%
with 4-ddma-atc and to 58% with atc, thus showing
only fivefold improved repression with 4-ddma-atc over
atc. A similar result is found for TetR
i2
-L17V V20F,
while the single exchange mutant TetR
i2
-N18Y shows
no distinction between atc and 4-ddma-atc.
Randomization of helix a4 also led to three candi-
dates. The amino-acid exchanges I59L L60M yield a
TetR

i2
mutant with reduced inducibility. TetR
i2
-I59V
H63Q exhibits a reverse phenotype but only little
specificity with fourfold increased corepression with
4-ddma-atc over atc. The I59S exchange leads to
repression of about 20% with 4-ddma-atc and to
about 50% with atc. While the efficiency is not great,
this mutation nevertheless proves that exchange of a
single amino acid can be sufficient to reverse the induc-
tion properties of TetR
i2
.
The pool with the randomizations in helix a6 yielded
nine candidates. They contain single, double and triple
mutations conferring 4-ddma-atc specific reverse phe-
notypes, and all of them were confirmed in liquid cul-
ture. The single amino-acid exchanges D95H, G96V or
K98N yield revTetR
i2
mutants. The G96V exchange
leads to an about 20-fold increased repression with
4-ddma-atc compared to atc. It is noteworthy that this
mutation does not change the property of wild-type
TetR [8]. Therefore, we asked if single residue muta-
tions leading to revTetR generally behave different
when introduced into TetR
i2
. As single exchange rev-

TetR variants were found for the residues V99 and
L17 [8] and V99G did reverse the TetR
i2
activity [9] we
analyzed the role of substitutions at these positions for
both effectors in the wild-type and TetR
i2
sequence
backgrounds.
TetR
i2
variants with mutations at valine 99
We revisited the 19 possible exchanges at positions 99
in TetR [8] and introduced them into TetR
i2
. The
in vivo repression and induction for these 20 TetR vari-
ants is shown in Fig. 2A,B. Fig. 2A shows the activit-
ies of V99 exchanges in TetR for the effectors atc and
4-ddma-atc and without effector. 11 out of 19 substitu-
tions at V99 do not lead to large changes of the phe-
notype. However, 11 out of the 19 exchanges show
slightly enhanced repression with 4-ddma-atc com-
pared to without thus making it a corepressor for these
variants. Interestingly, the mutations V99I, V99M,
V99P, V99G and V99F turn 4-ddma-atc into a core-
pressor while atc is still an inducer. This is remarkable
because a residue at position 99 is not in contact with
the effector [2]. Six out of these 11 exchanges exhibit a
reverse phenotype with 4-ddma-atc in the TetR

i2
back-
ground. Thus, 4-ddma-atc still acts as corepressor but
repression in the absence of effector is lost, despite of
the fact that a residue at position 99 does not contact
DNA, either [3]. Thirteen out of the 19 mutations in
TetR
i2
cause almost complete loss of repression with-
out effector, indicating that this mutant is generally
more sensitive for additional mutations. While substi-
tutions of V99 with the charged amino acids R, K or
E in wild-type TetR show pronounced reverse pheno-
types, all charged amino acids at this position in
TetR
i2
lead to only very weak reverse phenotypes with
4-ddma-atc (Fig. 2B).
Despite of their chemical similarity, serine and
threonine cause contrary effects when replacing V99:
TetR
i2
-V99S shows fivefold better repression with
4-ddma-atc, while V99T enhances repression with atc
sevenfold. Residues with aromatic side chains at posi-
tion 99 exhibit regulatory effects according to their
size: the V99W exchange shows a 1.5-fold, V99Y a
threefold, and V99F a 4.5-fold preference for 4-ddma-
atc over atc as corepressor. There is no relationship
between phenotype and size at this position in the

TetR background. The V99G exchange in TetR only
marginally influences induction with atc but leads to
Table 1. In vivo repression and induction of TetR
i2
variants. The
expression of 100% b-galactosidase corresponds to 6300 ± 1050
units.
TetR variant
b-Gal activity (%)
Induction with
4-ddma-atc
(0.4 l
M)
atc
(0.4 lM)
TetR
i2
1.6 ± 0.1 57 ± 5 3.1 ± 0.5
TetR
i2
-N18Y 60 ± 1.3 20 ± 1 14 ± 1.2
TetR
i2
-L17V V20F 80 ± 6 11 ± 0.5 34 ± 0.7
TetR
i2
-E15A L17V 85 ± 2 12.5 ± 0.4 58 ± 8
TetR
i2
-I59S 87 ± 1 19 ± 0.6 46 ± 1

TetR
i2
-I59V H63Q 84 ± 3 5 ± 4 20 ± 1
TetR
i2
-I59L L60M 8 ± 0.8 17 ± 0.9 3 ± 0.1
TetR
i2
-G96V 67 ± 0.9 2.4 ± 0.1 51 ± 4
TetR
i2
-D95H 58 ± 3.4 2 ± 0.1 4 ± 0.4
TetR
i2
-K98N 63 ± 1 6 ± 0.4 18 ± 2
TetR
i2
-R94C D95C 79 ± 8 2 ± 0.1 51 ± 5
TetR
i2
-D95A N81S 81 ± 2.7 2 ± 0.2 54 ± 4
TetR
i2
-Y93C V99M 80 ± 6 4 ± 0.2 8 ± 0.6
TetR
i2
-R94H K98I V99R 45 ± 3 1 ± 0.1 17 ± 0.5
TetR
i2
-R94S K98I H100Q 58 ± 3 4 ± 0.1 53 ± 3

TetR
i2
-R94H D95N G96R 60 ± 1.4 2 ± 0.1 4 ± 0.4
E M. Henßler et al. Altered TetR effector binding and allostery
FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS 4489
improved repression with 4-ddma-atc. The same
exchange leads to one of the best 4-ddma-atc specific
reverse phenotypes in TetR
i2
-V99G. Another very spe-
cific reverse phenotype is found for TetR
i2
-V99N with
a 34-fold better repression in the presence of 4-ddma-
atc compared to atc. Again, this exchange has only a
slight effect on induction with atc when introduced in
the TetR sequence background.
Western blot analyses of the V99A, V99G, V99S,
V99T and V99N exchanges in wild-type and TetR
i2
revealed only small differences of the intracellular pro-
tein amounts (Fig. 2C), which may correlate to the
alterations seen in the repressed expression levels of
the respective mutants. The observed specificity chan-
ges are clearly not influenced by protein amounts.
TetR
i2
variants with mutations at leucine 17
The effects of all possible exchanges of leucine at
position 17 in the wild-type or TetR

i2
sequence back-
grounds are shown in Fig. 3A,B. Most of the larger
and charged amino acids at this position lead to
repression deficient proteins in both sequence back-
grounds. TetR is less proned for activity loss due to
mutation at this position as 10 of 19 substitutions
lead to altered phenotypes, while 17 out of 19 substi-
tutions cause more or less severe loss of repression
without effector in TetR
i2
. We obtained eight variants
showing improved repression with 4-ddma-atc com-
pared to without, among them four exchanges where
atc is an inducer and 4-ddma-atc a corepressor. Five
out of these mutations cause 4-ddma-atc sensitive
reverse phenotypes. The TetR
i2
-L17M or -L17I
exchanges show increased repression with atc, but not
with 4-ddma-atc. Exchanges leading to 4-ddma-atc
dependent repression in TetR
i2
include the aromatic
amino acids W and Y, the hydroxyl containing resi-
dues S and T, and C. TetR
i2
-L17A is the best rev-
TetR
i2

showing repression to 3% with 4-ddma-atc
and only to 59% with atc. In contrast, TetR-L17A is
noninducible with atc or 4-ddma-atc, while TetR-
L17G shows the best atc dependent reverse pheno-
type. TetR
i2
-L17G, on the other hand, is inactive.
Taken together, it is surprising that single residue
exchanges cause quite different effects in these two
sequence backgrounds. Moreover, contrary activities
are caused by very small differences in side chains.
The determination of the intracellular protein
amounts (Fig. 3C) excludes contributions to these spe-
cificity changes.
Specificity determining residues in TetR
i2
-V99G
TetR
i2
-V99G is one of the best revTetR
i2
variants with
4-ddma-atc specific repression, almost completely lack-
ing repression with atc [9]. As the V99G exchange in
the wild-type sequence background displays no reverse
phenotype and slightly increased repression in the
presence of 4-ddma-atc, we decided to determine the
contribution of each amino-acid exchange in TetR
i2
-

V99G to the combined activity. We constructed and
B
C
A
Fig. 2. Regulatory properties of TetR variants with mutations at
position 99. b-Galactosidase activities of E. coli WH207 ⁄ ktet50
transformed with plasmids bearing either no tetR or different tetR
variants are shown. They were determined in the presence of
0.4 l
M atc (white columns), 0.4 lM 4-ddma-atc (grey) or in the
absence of effector (black). The b-Gal activity in the absence of
tetR was set to 100% and corresponds to 6300 ± 1050 units.
(A) Regulatory properties for the wild-type TetR sequence back-
ground with all mutations at Val99 and (B) for the TetR
i2
sequence
background with all possible mutations at Val99. (C) Steady-state
levels of selected TetR variants with mutations of V99. The first
lane (TetR) contains 50 ng of purified wild-type TetR and the other
lanes 50 lg of a soluble protein extract from E. coli WH207 ⁄ ktet50.
Altered TetR effector binding and allostery E M. Henßler et al.
4490 FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS
analyzed all possible double and triple mutants that
include the mutation V99G. The results obtained in
our E. coli indicator strain are shown in Fig. 4. As
TetR-H64K V99G S138I has nearly the same proper-
ties than the protein with all four exchanges and
TetR-V99G S135L is not reverse and atc specific, the
S135L mutation does not contribute to the TetR
i2

-
V99G activity profile. The mutants TetR-V99G S138I,
TetR-H64K V99G S135L, and TetR-H64K V99G
clearly have reverse activities, albeit with different effi-
ciencies, but do not show the effector specificity.
TetR-V99G S135L S138I is inactive. These results
demonstrate that both the H64K and S138I mutations
are necessary in combination with V99G to produce
revTetR variants with 4-ddma-atc specificity. It is also
remakable that the S138I mutation does not always
lead to 4-ddma-atc specificity as seen in TetR-V99G
S138I, however, this mutant is only slightly reverse
with both effectors.
Thermodynamic analysis of atc and 4-ddma-atc
binding to TetR
i2
-L17A and TetR
i2
-V99N
For overexpression of the proteins the respective genes
were introduced into pWH610 and the resulting plas-
mids were transformed in E. coli RB791 [11]. TetR
i2
-
L17A was purified to homogeneity employing the
protocol described for wild-type TetR [11]. The purifi-
cation protocol for TetR
i2
-V99N had to be modified
as described in experimental procedures and resulted

in a protein with 50% activity. To quantify atc and
4-ddma-atc binding to the TetR variants we titrated
0.005 lm, 0.01 lm or 0.1 lm atc or 4-ddma-atc with
each TetR protein in fluorescence buffer containing
20 mm MgCl
2
. Under these conditions, binding of the
Fig. 4. Contribution of mutations H64K, S135L and S138I to the
activity of TetR
i2
-V99G. b-Gal activities were measured in E. coli
WH207 ⁄ ktet50 transformed with a plasmid bearing either no tetR
or different tetR variants. b-Gal activities are shown in the presence
of 0.4 l
M atc (white columns) or 4-ddma-atc (grey) or without effec-
tor (black). The combination of mutations is indicated at the bottom
of the figure. b-Gal activity in the absence of tetR was set to 100%
and corresponds to 6300 ± 1050 units.
A
B
C
Fig. 3. Regulatory properties of TetR variants with mutations at
position 17. b-Gal activities of E. coli WH207 ⁄ ktet50 transformed
with plasmids bearing either no tetR or different tetR variants are
shown. They were determined in the presence of 0.4 l
M atc (white
columns), 0.4 l
M 4-ddma-atc (grey) or without effector (black).
b-Gal activity of 100% corresponds to 6300 ± 1050 units. (A)
Results are shown for all possible residues at position 17 in the

wild-type TetR sequence background and (B) for the TetR
i2
sequence background. (C) Steady-state levels of selected TetR vari-
ants with mutations of L17. The first lane (TetR) contains 50 ng of
purified wild-type TetR and the other lanes 50 lg of a soluble pro-
tein extract from E. coli WH207 ⁄ ktet50.
E M. Henßler et al. Altered TetR effector binding and allostery
FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS 4491
[atc-Mg]
+
or [4-ddma-atc-Mg]
+
complex to TetR can
be directly monitored. Atc or 4-ddma-atc fluorescence
were employed to observe complex formation. The fits
of the data for all TetR variants indicated positive
cooperativity. As described previously for tetracycline
and atc [12,13], we observed only weak cooperativity
for binding of 4-ddma-atc to the wild-type TetR and
of atc to TetR
i2
. In contrast, 4-ddma-atc binding to
the revTetR
i2
variants showed large cooperativity.
Scatchard analysis confirmed positive cooperativity for
TetR
i2
-L17A and -V99N (Fig. 5). The resulting equi-
librium binding constants are summarized in Table 2.

As we could not saturate atc binding to TetR
i2
-L17A
and -V99N at 5 lm of atc, we assume constants below
2 · 10
5
m
)1
. The equilibrium binding constant of atc
to TetR
i2
was determined previously by Mg
2+
-depend-
ent titrations [5] and yielded a K
A
of 1.7 · 10
7
m
)1
.
The direct titration used here uncovers weak coopera-
tivity (a sixfold higher affinity for binding to the sec-
ond atc) but the constants are in the same range.
Binding of the first and the second 4-ddma-atc to
wild-type TetR are roughly 10-fold higher than deter-
mined previously [5]. Both revTetR
i2
variants exhibit
higher affinities for 4-ddma-atc compared to atc but

the affinities are lower than the respective ones to
TetR
i2
.
Fig. 5. Binding curves and Scatchard plots
of binding of TetR to [4-ddma-atc-Mg]
+
.
Fluorescence titrations were carried out at
0.1 l
M, 0.01 lM and 0.005 lM [Atc-Mg]
+
.
m is the average number of 4-ddma-atc mole-
cules bound to one TetR monomer. The
circles show the data, and the lines indicate
the fit according to the binding function. The
nonlinear curve progression shows the pres-
ence of positive cooperativity for the two
4-ddma-atc binding sites. (A) Fluorescence
titration, Langmuir fit and Scatchard plot of
the titration of 4-ddma-atc with TetR
i2
-L17A.
(B) Fluorescence titration, Langmuir fit and
Scatchard plot of the titration of 4-ddma-atc
with TetR
i2
-V99N.
Table 2. Atc and 4-ddma-atc binding constants of TetR variants. All constants have been determined by direct titration of 0.1 lM, 0.01 lM or

0.005 l
M [atc-Mg]
+
or [4-ddma-atc-Mg]
+
with TetR and are compared to binding constants obtained previously by titration at limiting MgCl
2
concentrations [5]. TetR
M
corresponds to one monomer which can bind one [tc-Mg]
+
.TetR
D
represents the dimer that binds [tc-Mg]
+
which
can then bind the second molecule.
Equilibrium binding constants
a
,(·10
7
M
)1
)
[4-ddma-atc-Mg]
+
[atc-Mg]
+
TetR TetR
i2

TetR
i2
-V99N
TetR
i2
-L17A TetR TetR
i2
TetR
i2
-V99N
TetR
i2
-L17A
TetR
M
+ [tc-Mg]
+
?TetR
M
[tc-Mg]
+
0.3
b
132
b
119600
b
1.7
b
< 0.02

c
< 0.02
c
TetR
D
+ [tc-Mg]
+
? TetR
D
[tc-Mg]
+
1.5 –
d
0.1 0.4 –
d
1.2 < 0.02
c
< 0.02
c
TetR
D
[tc-Mg]
+
+ [tc-Mg]
+
? TetR
D
[tc-Mg]
+
2

6.3 –
d
22 31 –
d
3.7 < 0.02
c
< 0.02
c
a
The standard deviations typically range from 10% to 40%.
b
See [5].
c
The affinity was too low to be quantified.
d
The affinity was too high
for quantification by direct titrations.
Altered TetR effector binding and allostery E M. Henßler et al.
4492 FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS
TetO binding in the presence of both effectors was
qualitatively analyzed by EMSA for the revTetR
i2
vari-
ants. We used an at least 55-fold excess of 4-ddma-atc
or atc over TetR to ensure complete complex forma-
tion. The results are shown in Fig. 6. Both proteins
exhibit residual binding to tetO without effector and
with atc. The strongest tetO binding is observed for
TetR
i2

-V99N with 4-ddma-atc as corepressor, while
TetR
i2
-L17A does not show a clear specificity for one
of the effectors in this experiment.
Discussion
The two activities of TetR, DNA recognition and
effector binding, albeit located in different parts of the
protein, are connected by allostery and can be either
mutually exclusive (wild-type) or additive (revTetR)
[8]. Changes of DNA recognition specificities were
accomplished by mutations in the DNA reading head
[14,15] while alterations of effector specificity require
mutations near the respective binding pocket [5,6], and
changes of allostery can be accomplished by exchan-
ging residues in the contacting area between the DNA
reading head and the core of TetR (Fig. 1) [7,8]. In
addition to these structurally obvious location-function
relationships, the data presented here establish that
alterations of residues in that interface built by helices
a1, a4 and a6 (Fig. 1A) also affect both substrate
recognition properties, although they are located far
away from either binding site.
The same mutations in the wild-type TetR or TetR
i2
sequence backgrounds can lead to completely different
results, e.g. V99G in the wild-type is induced by atc
while the repression is increased in the presence of
4-ddma-atc, but DNA binding without effector is not
altered. In the TetR

i2
mutant, however, the same
exchange leads to loss of DNA binding in the absence
of effector and 4-ddma-atc dependent corepression.
Moreover, analysis of the contributions of each
exchange in TetR
i2
-V99G to effector specificity
revealed a similar role for V99G and S135L. It was
shown for S135L previously that it confers relaxed
effector specificity to TetR [5,6]. S135 belongs to the
secondary shell of the effector binding pocket which
does not directly contact tetracycline in the crystal
structure [2] but is located next to tc contacting resi-
dues. V99 is not in contact with S135 (Fig. 1B). Thus,
the effect of V99G on effector binding must be trans-
ferred to the effector binding pocket. V99A also leads
to similar properties as it has no effect on the wild-type
but shows corepression with atc and 4-ddma-atc in
TetR
i2
. V99S has unaltered properties in the wild-type
TetR, but leads to loss of DNA binding and 4-ddma-
atc dependent corepression in TetR
i2
. V99T, on the
other hand, shows partial DNA binding, corepression
by atc and induction by 4-ddma-atc when introduced
in TetR
i2

. Thus, the addition of a single methyl group-
ing has remarkable differential effects on the activities
of these two TetR variants. A similar result is also
observed for exchanges at position 17. L17C, F, I and
V alter the allostery of TetR
i2
while the wild-type activ-
ity is not affected. Exchanges of L17 for A, T, W or Y
influence allostery and effector recognition.
The thermodynamic analysis of TetR
i2
-L17A and
-V99N revealed reduced binding constants for 4-ddma-
atc and atc. Thus, the in vivo effects reflect large affin-
ity changes reinforcing clear structural influence of
these mutations on the effector binding pocket. More-
over, the wild-type TetR has no apparent cooperativity
for effector binding [12] yet we observed positive coop-
erativity for the TetR mutants. Cooperativity has been
described for IPTG binding to the Lac repressor-oper-
ator DNA complex [16] and for tetracycline binding to
the TetR-tetO complex [17] but binding to both free
proteins is not cooperative [12,18]. Thus, it seems that
not only the affinity but also the nature of effector
recognition may be altered by the mutations studied
here.
The structural details underlying these long range
effects are not clear at present. It has been proposed that
the reduced effector binding affinities for revTetR-G96E
Fig. 6. EMSA of tet operator with TetR

i2
-V99N and TetR
i2
-L17A.
The EMSA was performed without effector and in the presence of
0.1 m
M atc or 4-ddma-atc. Hybridized oligonucleotides (0.3 lM) car-
rying tetO or a nonpalindromic sequence (usp. DNA) were incuba-
ted for 15 min with 0.3 l
M,0.9lM or 1.8 lM of the respective
TetR variant, electrophoresed on an 8% polyacylamide gel and
stained with ethidium bromide. The contents of the mixtures ana-
lyzed are indicated below the respective slots.
E M. Henßler et al. Altered TetR effector binding and allostery
FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS 4493
L205S may be due to changes in the positioning of the
effector contacting residue H100 [7]. This assumption
could apply to the properties of TetR
i2
-V99N, but the
exchanged residue in TetR
i2
-L17A is not in proximity to
H100 or to any other residue contacting the effector [2].
In conclusion, we propose that revTetR mutations
do not only lead to the previously proposed reposition-
ing of the DNA reading heads with respect to the core
domain [7,8] but also to altered effector binding via
structural changes in the effector binding pocket. It
was proposed on the basis of the TetR crystal struc-

tures [2–4,19] that the structural changes upon effector
binding are transmitted through the protein to the
DNA-binding head via the interface region. As a result
the distance between the two recognition helices is
increased, leading to loss of DNA binding. Helices a1,
a4 and a6 forming this interface are involved in signal
transduction, but there is no structural hint for an
influence on effector binding. As we clearly demon-
strate specificity effects of residues in this region on
effector binding [20] this must be an as of yet unrecog-
nized contribution of effector binding site flexibility to
TetR allostery.
Experimental procedures
Materials and general methods
Atc was from Acros (Geel, Belgium) and 4-ddma-atc was
synthesized by Susanne Lochner and Peter Gmeiner (Phar-
mazeutische Chemie, FAU Erlangen-Nu
¨
rnberg). All other
chemicals were from Merck (Darmstadt, Germany), Roth
(Karlsruhe, Germany) or Sigma (Munich, Germany).
Enzymes for DNA restriction and modification were from
New England Biolabs (Frankfurt ⁄ Main, Germany), Roche
(Mannheim, Germany), Stratagene (Heidelberg, Germany)
or Pharmacia (Freiburg, Germany). Oligonucleotides were
purchased from MWG Biotech (Ebersberg, Germany).
Isolation and manipulation of DNA was performed as
described previously [21].
Construction of the TetR mutant pools
Escherichia coli DH5a was used for cloning. The DNA con-

taining randomized codons for helices a1 and a6 from
pWH1925 [8] were introduced in pWH1925-tetR
i2
(enco-
ding the mutations H64K S135L S138I) via XbaI ⁄ ApaI and
ApaI ⁄ FspI, respectively. Randomization of codons 50–63
in helix a4 was performed by PCR mutagenesis with the
primers a4deg_H64K (5¢-aataagcgggcccta
ctggatgcgctggcggt
ggagatcttggcgcgtcataaggattat-3¢; the underlined positions
contain 89% wild-type and 11% of the three non-wt bases
resulting in a predominant frequency of three to four muta-
tions) and 1925gh (5¢-gcaaaccgcctctcgccgc-3¢) using tetR
i2
as template. The resulting fragment was introduced in
pWH1925 via ApaI ⁄ NcoI for constitutive expression. All
other TetR variants were constructed using single restric-
tion enzyme sites in pWH1925.
E. coli screening system
E. coli WH207 ⁄ ktet50 [10,22] was transformed with the
mutant pools. It contains a chromosomal tetA-lacZ fusion
under tetR control. The cells were plated on MacConkey
Agar Base (Becton Dickinson, San Jose, CA, USA) con-
taining 14 gÆL
)1
lactose, 0.0042% (w ⁄ v) neutral red and
0.0014% (w ⁄ v) crystal violet. The colonies were screened
for their ability to repress b-galactosidase in the presence of
0.4 lm 4-ddma-atc and to express b-galactosidase on plates
containing 0.4 lm atc.

b-Galactosidase assays
Repression and induction with different tc analogs was
determined in E. coli WH207 ⁄ ktet50. Cells were grown in
LB supplemented with 0.4 lm of atc or 4-ddma-atc at
37 °C. b-Galactosidase activities were determined as des-
cribed [23]. Three independent cultures were assayed for
each mutant and measurements were repeated at least twice.
The expression of b-galactosidase in the absence of TetR
was set to 100% and corresponds to 6300 ± 1050 units.
Protein purification
E. coli RB791 was transformed with pWH610 containing
the respective mutations. Purification of TetR
i2
-L17A to
homogeneity was performed as described [11]. For purifica-
tion of TetR
i2
-V99N the E. coli cells were resuspended in
50 mm Na-phosphate pH 6.8, 50 mm NaCl, 25% (w ⁄ v)
sucrose, 1 mm EDTA and 10 mm dithiothreitol. Cell dis-
ruption was achieved by sonification following addition of
5 mg lysozyme, 0.25 mg DNaseI, 2 mm MgCl
2
,1%(v⁄ v)
Triton X-100 and 1% (w ⁄ v) Na-deoxycholate and incuba-
tion for 30 min at room temperature. The suspension was
frozen in liquid N
2
after adjustment to 6 mm EDTA and
thawed at 37 °C. The protein was purified from the super-

natant by cation exchange and size exclusion chromatogra-
phy as described previously [11].
The protein concentrations were determined by UV
spectroscopy and their activity was assessed by saturating
titration with 4-ddma-atc observing the change of fluores-
cence.
Fluorescence measurements
The fluorescence measurements were performed in a Spex
Fluorolog 3 with two double monochromators. To observe
Altered TetR effector binding and allostery E M. Henßler et al.
4494 FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS
4-ddma-atc fluorescence we excited at 420 nm and observed
emission at 540 nm. Excitation of atc fluorescence was per-
formed at 454 nm and emission was observed at 545 nm.
The equilibrium binding constants were obtained from
fluorescence titrations under equilibrium conditions. The
titrations were carried out in buffer containing 100 mm
Tris ⁄ HCl, pH 8.0, 100 mm NaCl and 20 mm MgCl
2
.
0.1 lm, 0.01 lm or 0.005 lm atc or 4-ddma-atc were titra-
ted with TetR concentrations from 2 · 10
)10
m to
1 · 10
)5
m. All measurements were repeated at least twice.
The binding constants were calculated by fitting of a hyper-
bolic binding function and including cooperative binding.
Electrophoretic mobility shift assay

The synthetic tetO1 containing fragment 5¢-gggtgtgcc
gacactctatcattgatagagttattatac-3¢ and tetO2 containing the
complementary sequence were used for EMSA. The TetR
recognition site is depicted in bold style. For hybridization,
equal molar amounts of each oligonucleotide were mixed in
water, heated at 94 °C for 2 min and allowed to cool down
to room temperature within 2 hÆ6 pmol of the DNA was
incubated with atc, 4-ddma-atc or without effector and the
indicated amounts of protein. An oligonucleotide contain-
ing no palindromic sequence was used as a negative control
(5¢-ctaataaaattaatcatttatggcataggcaacaag-3¢). All samples
were incubated in complex buffer containing 0.02 m
Tris ⁄ HCl (pH 8.0) and 5 mm MgCl
2
. Atc and 4-ddma-atc
were added to a final concentration of 0.1 mm. After incu-
bation for 15min at room temperature, the DNA was elec-
trophoresed on an 8% polyacylamide gel at 100 V in TBM
buffer containing 89 mm Tris, 89 mm boric acid and 1 m m
MgCl
2
. The DNA was detected by ethidium bromide
staining.
Acknowledgements
We thank Susanne Lochner and Prof. Peter Gmeiner
for kindly providing 4-ddma-atc and Dr. Oliver Scholz
for fruitful discussions.
This work was supported by the Deutsche Fors-
chungsgemeinschaft through SFB 473 and the Fonds
der Chemischen Industrie.

References
1 Berens C & Hillen W (2003) Gene regulation by tetra-
cyclines. Constraints of resistance regulation in bacteria
shape TetR for application in eukaryotes. Eur J Bio-
chem 270, 3109–3121.
2 Kisker C, Hinrichs W, Tovar K, Hillen W & Saenger WB
(1995) The complex formed between Tet repressor and
tetracycline-Mg2+ reveals mechanism of antibiotic
resistance. J Mol Biol 247, 260–280.
3 Orth P, Alings C, Schnappinger D, Saenger W & Hin-
richs W (1998) Crystallization and preliminary X-ray
analysis of the Tet-repressor ⁄ operator complex. Acta
Crystallogr D54, 99–100.
4 Orth P, Schnappinger D, Sum PE, Ellestad GA,
Hillen W, Saenger W & Hinrichs W (1999) Crystal
structure of the Tet repressor in complex with a novel
tetracycline, 9-(N,N-dimethylglycylamido)-6-demethyl-
6-deoxy-tetracycline. J Mol Biol 285, 455–461.
5 Henssler EM, Scholz O, Lochner S, Gmeiner P & Hillen
W (2004) Structure-based design of tet repressor to opti-
mize a new inducer specificity. Biochemistry 43, 9512–
9518.
6 Scholz O, Ko
¨
stner M, Reich M, Gastiger S & Hillen
WB (2003) Teaching TetR to recognize a new inducer.
J Mol Biol 329, 217–227.
7 Kamionka A, Bogdanska-Urbaniak J, Scholz O &
Hillen W (2004) Two mutations in the tetracycline
repressor change the inducer anhydrotetracycline to a

corepressor. Nucleic Acids Res 32, 842–847.
8 Scholz O, Henssler EM, Bail J, Schubert P, Bogdanska-
Urbaniak J, Sopp S, Reich M, Wisshak S, Ko
¨
stner M,
Bertram R & Hillen W (2004) Activity reversal of Tet
repressor caused by single amino acid exchanges. Mol
Microbiol 53, 777–789.
9 Bertram R, Kraft C, Wisshak S, Mu
¨
ller J, Scholz O
& Hillen W (2005) Phenotypes of combined Tet
repressor mutants for effector and operator recogni-
tion and allostery. J Mol Microbiol Biotechnol 8,
104–110.
10 Wissmann A, Wray LV Jr, Somaggio U, Baumeister R,
Geissendo
¨
rfer M & Hillen W (1991) Selection for Tn10
Tet repressor binding to tet operator in Escherichia coli:
isolation of temperature-sensitive mutants and combina-
torial mutagenesis in the DNA binding motif. Genetics
128, 225–232.
11 Ettner N, Muller G, Berens C, Backes H, Schnappinger
D, Schreppel T, Pfleiderer K & Hillen WB (1996) Fast
large-scale purification of tetracycline repressor variants
from overproducing Escherichia coli strains. J Chroma-
togr A 742, 95–105.
12 Takahashi M, Altschmied L & Hillen W (1986) Kinetic
and equilibrium characterization of the Tet repressor-

tetracycline complex by fluorescence measurements.
Evidence for divalent metal ion requirement and energy
transfer. J Mol Biol 187, 341–348.
13 Scholz O, Kintrup M, Reich M & Hillen WB (2001)
Mechanism of Tet repressor induction by tetracyclines:
length compensates for sequence in the alpha8-alpha9
loop. J Mol Biol 310, 979–986.
14 Helbl V & Hillen W (1998) Stepwise selection of TetR
variants recognizing tet operator 4C with high affinity
and specificity. J Mol Biol 276, 313–318.
15 Helbl V, Tiebel B & Hillen W (1998) Stepwise selec-
tion of TetR variants recognizing tet operator 6C
E M. Henßler et al. Altered TetR effector binding and allostery
FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS 4495
with high affinity and specificity. J Mol Biol 276,
319–324.
16 O’Gorman RB, Rosenberg JM, Kallai OB, Dickerson
RE, Itakura K, Riggs AD & Matthews KS (1980)
Equilibrium binding of inducer to lac repressor.
operator DNA complex. J Biol Chem 255, 10107–
10114.
17 Lederer T, Takahashi M & Hillen W (1995) Thermo-
dynamic analysis of tetracycline-mediated induction of
Tet repressor by a quantitative methylation protection
assay. Anal Biochem 232, 190–196.
18 Friedman BE, Olson JS & Matthews KS (1977) Inter-
action of lac repressor with inducer, kinetic and equili-
brium measurements. J Mol Biol 111, 27–39.
19 Hinrichs W, Kisker C, Duvel M, Mu
¨

ller A, Tovar K,
Hillen W & Saenger W (1994) Structure of the Tet
repressor-tetracycline complex and regulation of anti-
biotic resistance. Science 264, 418–420.
20 Orth P, Cordes F, Schnappinger D, Hillen W, Saenger W
& Hinrichs W (1998) Conformational changes of the
Tet repressor induced by tetracycline trapping. J Mol Biol
279, 439–447.
21 Sambrook J (2001) Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, USA.
22 Smith LD & Bertrand KP (1988) Mutations in the Tn10
tet repressor that interfere with induction. Location of
the tetracycline-binding domain. J Mol Biol 203, 949–
959.
23 Miller JH (1972) Experiments in molecular genetics.
Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, USA.
Altered TetR effector binding and allostery E M. Henßler et al.
4496 FEBS Journal 272 (2005) 4487–4496 ª 2005 FEBS