Structural origins for selectivity and specificity in an
engineered bacterial repressor–inducer pair
Michael A. Klieber
1
, Oliver Scholz
2,
*, Susanne Lochner
3
, Peter Gmeiner
3
, Wolfgang Hillen
2
and Yves A. Muller
1
1 Lehrstuhl fu
¨
r Biotechnik, Department of Biology, Friedrich-Alexander University, Erlangen-Nuremberg, Germany
2 Lehrstuhl fu
¨
r Mikrobiologie, Department of Biology, Friedrich-Alexander University, Erlangen-Nuremberg, Germany
3 Lehrstuhl fu
¨
r Pharmazeutische Chemie, Department of Chemistry and Pharmacy, Friedrich-Alexander University, Erlangen-Nuremberg,
Germany
Introduction
The bacterial repression system consisting of the effec-
tor molecule tetracycline, the tetracycline-inducible
repressor protein tetracycline repressor (TetR) and the
tet operator (tetO) has proven itself to comprise a
valuable tool for studying gene expression not only in
prokaryotes, but also in eukaryotes [1–3]. The repres-

for the 4-ddma-atc complex
(Received 10 March 2009, revised 9 July
2009, accepted 31 July 2009)
doi:10.1111/j.1742-4658.2009.07254.x
The bacterial tetracycline transcription regulation system mediated by the
tetracycline repressor (TetR) is widely used to study gene expression in
prokaryotes and eukaryotes. To study multiple genes in parallel, a triple
mutant TetR(K
64
L
135
I
138
) has been engineered that is selectively induced
by the synthetic tetracycline derivative 4-de-dimethylamino-anhydrotetracy-
cline (4-ddma-atc) and no longer by tetracycline, the inducer of wild-type
TetR. In the present study, we report the crystal structure of
TetR(K
64
L
135
I
138
) in the absence and in complex with 4-ddma-atc at reso-
lutions of 2.1 A
˚
. Analysis of the structures in light of the available binding
data and previously reported TetR complexes allows for a dissection of the
origins of selectivity and specificity. In all crystal structures solved to date,
the ligand-binding position, as well as the positioning of the residues lining

64
L
135
I
138
) in which residues His64,
Ser135 and Ser138 of TetR have been replaced by Lys,
Leu and Ile, respectively [9]. This mutant is selectively
induced by the synthetic inducer 4-de-dimethylamino-
anhydrotetracycline (4-ddma-atc) and slightly by atc,
but no longer by tetracycline. To better understand the
switch in selectivity and the acquired novel specificity
of TetR(K
64
L
135
I
138
) for 4-ddma-atc, we solved the
crystal structure of TetR(K
64
L
135
I
138
) in the presence
and absence of 4-ddma-atc at resolutions of 2.06 and
2.1 A
˚
, respectively. We show that the effects of the

both monomers.
The overall structures of ligand-free TetR(K
64
L
135
I
138
) and of TetR(K
64
L
135
I
138
) in complex with
4-ddma-atc are very similar (for the chemical structure
of 4-ddma-atc and related TetR ligands, see Fig. 2).
The two crystals that have been used for structure
determination are highly isomorphous and only small
deviations occur in the cell axes (Table 1). They each
contain a complete dimer in the asymmetric unit.
Almost no differences exist between the monomers in
each crystal and the monomers ⁄ dimers between crys-
tals. The main chain atoms of the two monomers in
each crystal can be superimposed with rmsd of 0.92
and 1.34 A
˚
for the 4-ddma-atc-bound and effector-free
TetR(K
64
L

ligand. However, when considering the molecular
mechanism by which TetR exerts its function, then the
structural similarity might be considered unexpected.
A central function of TetR is its ability to adopt
different conformations. According to the conforma-
tional switch model, TetR exists in two conforma-
tions. In the ligand-free structure, the DNA-binding
heads are oriented such that TetR can readily bind
to the operator DNA, whereas, in effector-bound
TetR, the separation of the DNA-binding domain is
changed, such that TetR can no longer recognize the
tetO DNA sequence (Fig. 1A) [4]. However, as noted
above, the domain orientations in the 4-ddma-atc-
bound TetR(K
64
L
135
I
138
) and the ligand-free TetR
(K
64
L
135
I
138
) structure are very similar and resemble
that of induced TetR more closely than that of indu-
cer-free TetR (data not shown). Moreover, the main
chain of loop segment 100–105 that switches con-

I
138
). In this model,
induction can be explained by a shift of the population
towards a single DNA-binding incompetent conforma-
tion. Indications that such a population shift model
might apply to TetR have recently started to emerge
[13,14].
The effector-binding site of TetR(K
64
L
135
I
138
)in
the presence and absence of 4-ddma-atc
Particularly interesting with respect to the observed
specificity and selectivity of TetR(K
64
L
135
I
138
) for
4-ddma-atc are the interactions between the ligand
and the protein in the effector-binding site (Figs 1B
and 2A). Of the two binding sites that can be observed
independently in the 4-ddma-atc-complex structure,
A
C

) in complex with 4-ddma-atc (shown in magenta and yellow) superimposed onto the effector-free
TetR(K
64
L
135
I
138
) structure (shown in grey). Of the two binding sites present in the crystal structure, binding site I (Table 2) is shown. The
binding sites are highly similar in the presence and absence of 4-ddma-atc. The loop segment 100–105 that switches conformations in other
ligand-free and ligand-bound TetR structures is only slightly displaced in ligand-free TetR(K
64
L
135
I
138
) compared to the ligand-bound TetR
structure.
Structure of an engineered TetR-inducer pair M. A. Klieber et al.
5612 FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS
both binding sites show the density for the ligand in
the initial difference-fourier electron density maps,
albeit to a different extent. The ligand is well defined
in binding site I, but only poor density has been
observed for the ligand in binding site II. To account
for this observation, the occupancy of the ligand in
binding site II was estimated at 50% and that in bind-
ing site I at 100%. When using these values during the
refinement, the temperature factors of the ligands
refine to values similar to those of the surrounding res-
idues, hinting that the estimated occupancies correctly

ligand in the present study is restricted to 4-ddma-atc
binding to site I in TetR(K
64
L
135
I
138
).
4-ddma-atc binding leads to only minor rearrange-
ments in the TetR(K
64
L
135
I
138
)-binding site (Fig. 1C).
Among the most notable changes are a slight shift of the
entire loop segment 100–105 in the direction of the
ligand, the presence of two alternative side chain confor-
mations for Asn82 in the 4-ddma-atc-bound structure
versus a single conformation in ligand-free
TetR(K
64
L
135
I
138
) and, finally, the occurrence of a
slightly different rotamer for Ile138 (i.e. different posi-
tioning of atom Cd) in the ligand-free and ligand-bound

I
138
) binds 4-ddma-atc via a number of
specific interactions (Fig. 3A). One of the most notable
ones involves Lys64. Atom Nf of Lys64 interacts with
two oxygen atoms of 4-ddma-atc, namely of the amide
group attached to atom C2 and the OH group
A
B
C
D
Fig. 2. Chemical structures of (A) 4-ddma-atc, (B) atc, (C) tetracy-
cline (tc) and (D) dox.
M. A. Klieber et al. Structure of an engineered TetR-inducer pair
FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5613
attached to atom C3 of 4-ddma-atc. Furthermore,
atom Lys64-Nf is located in hydrogen-bonding dis-
tance to the amide group of Asn82 and the main chain
carbonyl oxygen atom of Tyr66. It is not possible to
predict the strength of the interaction between Lys64
and 4-ddma-atc based on structural data only. For
Table 1. Data collection and refinement statistics.
Triple mutant
TetR(K
64
L
135
I
138
)

2
) 15.1 26.5
Refinement statistics
Number of protein atoms, solvent
molecules and ligand atoms
3095, 116, 0 3180, 225, 58
R
work
(%) 21.6 20.3
R
free
(%) 26.2 26.1
rmsd bond lengths (A
˚
) 0.005 0.011
rmsd bond angles (°) 1.069 1.175
rmsd B-factors bonded atoms: main chain,
side chains (A
˚
2
)
1.51, 2.27 1.24, 2.13
Percentage of residues in most favored regions,
additional allowed, generously allowed and
disallowed regions of the Ramachandran plot
b
95.1, 4.9, 0.0, 0.0 95.5, 4.2, 0.3, 0.0
Average B-factor (A
˚
2

135
I
138
)I
a
4-ddma-atc
TetR
(K
64
L
135
I
138
)II
atc revTetR
(PDB code:
2VKV)
tc TetR(D)
(PDB code:
2VKE)
dox TetR(D)
(PDB code:
2O7O)
No ligand
TetR(K
64
L
135
I
138

– – 1.048
c
– 0.489, 1.062 0.432, 1.065 0.415, 0.999
Atc revTetR – – 0.431 0.896 – 0.413, 0.983 0.437, 0.918
tc TetR(BD) – – 0.456 1.024 0.270 – 0.216, 0.732
Dox TetR(BD) – – 0.523 0.878 0.259 0.265 –
a
Because the crystals contain two molecules in the asymmetric unit, two separate binding sites (I and II) are present in each of the two
TetR(K
64
L
135
I
138
) crystal structures.
b
Above the diagonal the rmsd (A
˚
) between structures of a selection of 93 residues surrounding the
ligand-binding site is reported (first number, rmsd obtained upon superposition of all main chain atoms of the selection; second number,
superposition of all atoms).
c
Below the diagonal: rmsd (A
˚
) calculated between the ligands (27 common ligand atoms) in the different com-
plexes after optimal superposition of the structures based on the main chain atoms of 93 residues surrounding the binding site.
Structure of an engineered TetR-inducer pair M. A. Klieber et al.
5614 FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS
example, the structure does not allow a distinction of
whether the side chain of Lys64 is protonated or not.

contact with the ligand at the ‘backside’ of 4-ddma-
atc. 4-ddma-atc directly contacts the mutated resi-
dues 135 (Ser135Leu) and 138 (Ser138Ile) of
TetR(K
64
L
135
I
138
). Because of the hydrophobic nature
of the Leu and Ile side chains and because of their
increased size compared to the serine residues in
A
C
B
Fig. 3. (A) Close-up view on the ligand-binding site of 4-ddma-atc in complex with TetR(K
64
L
135
I
138
) and (B) tetracycline in complex with TetR
[15] (PDB entry: 2VKE). The residues that differ between the two structures are underlined. The hydrogen-bonding network in which residue
64, namely Lys64 in TetR(K
64
L
135
I
138
) (A) and His64 in TetR (B), participates, is indicated by dashed lines. Water molecules present in the

with 4-ddma-atc without the addition of any extra
magnesium, a partially occupied magnesium ion can be
observed at a position identical to that observed in other
TetR effector complexes. With the exception of the
Lys64 interaction and the extended hydrophobic inter-
face introduced by Leu135 and Ile138, all other ligand
protein interactions are highly similar to those observed
in other TetR-ligand complexes (see also below).
Discussion
4-ddma-atc binding to TetR(K
64
L
135
I
138
) compared
to tetracycline, atc and dox binding to TetR
Various crystal structures of TetR in complex with tet-
racycline, dox and atc have already been solved to
high resolution. Comparing these structures among
themselves and to TetR(K
64
L
135
I
138
) promises to pro-
vide insight into why TetR(K
64
L

)in
complex with 4-ddma-atc and all other effector TetR
complexes is seen for the interaction between residue
64 and the various effectors. As noted above, in
TetR(K
64
L
135
I
138
) Lys64 is involved in a number of
specific interactions with 4-ddma-atc and it appears
that, in the wild-type TetR complexes, histidine is able
to participate in similar interactions because the posi-
tion of atom Ne of His64 coincides almost exactly with
that of atom Nf of Lys64 (atom displacement of
0.9 A
˚
; Fig. 3C). Compared to Lys64, however, a histi-
dine residue can participate in fewer hydrogen bonds.
In wild-type TetR, it appears that a putative hydrogen
atom attached to Ne of His64 is poised to interact
with the oxygen atom attached to atom C3 present in
all tetracycline derivatives. This oxygen is positioned in
plane with the imidazole ring, and a linear almost ideal
hydrogen bond can be anticipated for this interaction.
In comparison, an additional interaction often dis-
cussed as being important for ligand binding [16],
namely the interaction between Ne of His64 and the
amide group attached to atom C2 of tetracycline,

TetR(K
64
L
135
I
138
), where Ser135 and Ser138 are
replaced by Leu and Ile, water molecules are attached
to the serines in all other TetR structures and fill a
cleft between the ligand and the protein. Close inspec-
tion of these water molecules shows that their posi-
tions are largely conserved in the complexes formed
between TetR and tetracycline, dox or atc (Fig. 3C).
The specificity and selectivity of the 4-ddma-atc
TetR(K
64
L
135
I
138
) interaction
The structural investigations reported in the present
study aimed to gain insight into the mechanism
by which effector selectivity is switched in
TetR(K
64
L
135
I
138

pared to wild-type TetR (Table 3). In the case of the
ligands atc and 4-ddma-atc, only near additivity is
achieved in the mutants (7.93 versus 6.61 kcalÆmol
)1
for atc and )5.71 versus )3.60 kcalÆmol
)1
for 4-ddma-
atc binding).
In many cases, it is also possible to formulate almost
perfect thermodynamic cycles. For example, the free
binding energy difference observed for the binding of
the ligands atc and 4-ddma-atc to wild-type TetR
(DDG = 7.63 kcalÆmol
)1
) corresponds exactly to the
sum of the changes observed for atc binding to the
mutant His64Lys (4.7 kcalÆmol
)1
), the difference in
binding energies for the ligands atc and 4-ddma-atc to
the same mutant (1.28 kcalÆmol
)1
) and the differ-
ence in binding energies observed for 4-ddma-atc bind-
ing to wild-type TetR and to the His64Lys mutant
(1.65 kcalÆmol
)1
).
Juxtaposition of the binding affinities to the induc-
tion efficiencies suggests that free binding energies in

estimated to be in the range 10–40% of the reported
values [9]. When translated to DG, this corresponds to
approximately 0.25 kcalÆmol
)1
(Table 3).
The structures that we have determined in the pres-
ent study and the comparison of these structures with
previously solved crystal structures are in agreement
with the proposed additivity or near additivity of the
free binding energies. In all the structures, the effector
molecule binds at almost exactly the same position,
and the introduction of mutations and ⁄ or changes in
the ligand does not lead to any notable changes in the
side chain or backbone conformations of the residues
lining the binding site. Although the structures of each
single and double mutant have not been solved, it is
reasonable to assume that structure conservation also
extends to these mutants. As a result of the structural
Table 3. Induction efficiencies and free binding energies of TetR and mutants for various tetracycline analogs. Data are compiled from
Henssler et al. [9].
dox atc 4-ddma-atc
Induction efficiencies
TetR wild-type ++++
a
++++ )
H64K )))
S138I ) ++ )
S135L ++++ +++ +
H64K S138I )))
S135L S138I +++ +++ )

Free binding energies derived from the experimentally determined
binding affinities reported in Henssler et al.[9] and calculated according to DG=)RT lnK (t = 298.15 °K). In parentheses: DDG=DG(mutant)
) DG(wild-type).
c
The standard deviations of the affinities reported in Henssler et al. [9] have been estimated to be in the range 10–40%.
Assuming a standard error propagation model with dDG=)RT (dK ⁄ K), this translates into 0.05–0.25 kcalÆmol
)1
as an error estimate for DG.
M. A. Klieber et al. Structure of an engineered TetR-inducer pair
FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5617
conservation and the near additivity of the DG values,
it is possible to discuss the observed selectivity in light
of individual changes introduced in the binding site by
the various mutations.
With respect to single changes, the most drastic dif-
ferences in the free binding energies are observed for
the His64Lys mutation and for the removal of the
dimethyl-amino-group attached to atom C4 of the
tetracycline derivatives in wild-type TetR or in mutants
in which His64 is retained. Replacing the histidine by
a lysine causes all tetracycline derivatives to be recog-
nized with almost identical binding affinities (Table 3).
This is the result of a drastic decrease in the affinities
for dox and atc, and a significant increase in affinity for
4-ddma-atc. Because the free binding energies exceed
)12 kcalÆmol
)1
, the His64Lys mutation is not induced
by any of the four ligands anymore. Accordingly, it is
apparent that histidine is particular well suited to recog-

additional residue with 4-ddma-atc specificity [9].
Upon mutation of residue Ser135 to leucine, the
affinity of TetR for almost all ligands increases
(Table 3). This holds true for all the variants into
which this mutation is introduced. The only exception
appears to be the binding of dox to the single mutant
Ser135Leu for which a small decrease in affinity can
be observed compared to wild-type TetR
(DDG = +0.08 kcalÆmol
)1
). Introducing the mutation
Ser135Leu to any other variant also enhances the
binding affinity of dox. The amounts by which the
affinities increase differ for the various mutants and
the ligands. The most significant increase is observed
for 4-ddma-atc binding. Inspection of the crystal struc-
tures suggests that this increase in affinity is a direct
consequence of increased hydrophilic interactions and
the associated hydrophobic effect. As noted above, res-
idue 135 interacts with the largely hydrophobic D ring.
Whereas, in most crystal structures, a number of
highly conserved water molecules bridges between the
serine at position 135 and the tetracycline derivative,
the water molecules are expelled from this interface in
TetR(K
64
L
135
I
138

cine substitution leads to the observed shift in
specificities and no other hydrophobic residue is toler-
ated at this position [9]. The structure hints that an
isoleucine fits perfectly between the protein and the
tetracycline A and B rings.
The results obtained in the present study show
that the observed specificity and selectivity in
TetR(K
64
L
135
I
138
) for 4-ddma-atc can be explained
Structure of an engineered TetR-inducer pair M. A. Klieber et al.
5618 FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS
through a defined set of contributions. Whereas the
His64Lys mutation abolishes the selectivity present in
wild-type TetR, and the Ser135Leu mutation improves
the binding of all three effectors, the Ser138Ile muta-
tion selectively disfavors effector molecules containing
a dimethyl-amino group and, at the same time, only
slightly improves 4-ddma-atc binding. The physico-
chemical contributions of the individual residues
appear to be finely balanced and include geometrically
constrained hydrogen-bonding networks, electrostatic
interactions and solvation and dissolvation effects.
TetR(K
64
L

TetR variant D, is further modified through the introduc-
tion of three single site mutations, namely His64 fi Lys,
Ser135 fi Leu and Ser138 fi Ile. Transformed E. coli cells
were grown in LB medium at 28 °C and induced with
1mm isopropyl thio-b-d-galactoside after an A
600
of 0.8
was reached in the cell cultures. After further incubation
for 4 h, cells were harvested by centrifugation, and the pel-
let dissolved in 30 mL of 20 mm sodium phosphate buffer
(pH 6.8) containing 5 mm EDTA and 1 mm Pefabloc
(Roche Diagnostics, Mannheim, Germany) before the cell
walls were disrupted by sonication. After centrifugation
for 1 h at 100 000 g, the supernatant was purified employ-
ing a four-step chromatographic protocol. The supernatant
was first applied onto a weak cation-exchange column
(SP-Sepharose FF; GE Healthcare Bio-Sciences, Uppsala,
Sweden) and subsequently onto two strong anion-exchange
columns (Resource Q and Mono Q; GE Healthcare
Bio-Sciences). Whereas, in the case of the cation-exchange
column, the buffer was identical to the buffer used during
the sonication step, the two anion-exchange columns were
equilibrated with 50 mm NaCl, 20 mm Tris (pH 8.0). The
proteins were eluted from all three columns using a stan-
dard NaCl gradient (20 mm to 1 m). As a final chromato-
graphic step, a gel filtration chromatography run was
performed (Superdex 75; GE Healthcare Bio-Sciences)
using a buffer consisting of 200 mm NaCl and 50 m m Tris
(pH 8.0).
Initial crystallization conditions for TetR(K

138
) is not induced by tetra-
cycline [9]. Careful inspection of the initial and final elec-
tron density maps of the 4-ddma-atc-free TetR(K
64
L
135
I
138
)
structure did not provide any hints for the density of tetra-
cycline bound to the effector-binding site.
Crystals of TetR(K
64
L
135
I
138
) with 4-ddma-atc were
obtained upon soaking the previously grown ligand-free
crystals with a saturated solution of the poorly soluble
4-ddma-atc compound. The yellowish coloring of the crys-
tals indicated the successful incorporation of the ligand.
X-ray structure analysis and validation
Diffraction data sets of TetR(K
64
L
135
I
138

of 26.2% at 2.1 A
˚
(Table 1). The
M. A. Klieber et al. Structure of an engineered TetR-inducer pair
FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5619
determination of the ligand-bound TetR(K
64
L
135
I
138
) struc-
ture started with the coordinates of the ligand-free struc-
ture. Refinement of the complex converged at an R
work
of
20.7% and an R
free
value of 26.1%. The ligand 4-ddma-atc
and ligand restraints parameters were generated using
corina [26]. The individual models were validated using
the software procheck [27]. All structure illustrations were
prepared using pymol [28].
Structure comparisons
To investigate the origins of specificity and selectivity, the
structures of TetR(K
64
L
135
I

¨
ller from the Bessy
synchrotron Berlin during data collection, as well as
the anonymous reviewers for their valuable comments
and discussions. This work was supported through
funding from the Volkswagen foundation and DFG-
SFB473.
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