the V99G exchange in the wild-type leads to corepression by 4-ddma-atc without altering DNA binding.. However, in TetRi2 it leads to 4-ddma-atc dependent repression in combination with r
Trang 1Eva-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 (a5 to a10) 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 (TetRi2) 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: whillen@biologie.uni-erlangen.de
(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 TetRi2 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 TetRi2 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.
Trang 2was 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 TetRi2 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 TetRi2
As the most efficient and mechanistically most interest-ing mutations leadinterest-ing to revTetR occurred in helices a1, a4, and a6 [8], we decided to combine randomiza-tions in these helices with TetRi2(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-tetRi2 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-tetRi2 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, a4 and a6
(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).
Trang 3WH207⁄ 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 TetRi2-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 TetRi2-L17V V20F,
while the single exchange mutant TetRi2-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
TetRi2 mutant with reduced inducibility TetRi2-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 TetRi2
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 revTetRi2 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 TetRi2 As single exchange rev-TetR variants were found for the residues V99 and L17 [8] and V99G did reverse the TetRi2activity [9] we analyzed the role of substitutions at these positions for both effectors in the wild-type and TetRi2 sequence backgrounds
TetRi2variants with mutations at valine 99
We revisited the 19 possible exchanges at positions 99
in TetR [8] and introduced them into TetRi2 The
in vivorepression 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 TetRi2 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 TetRi2 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 TetRi2lead 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: TetRi2-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 4-ddma-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 l M )
TetR i2 -L17V V20F 80 ± 6 11 ± 0.5 34 ± 0.7
TetRi2-E15A L17V 85 ± 2 12.5 ± 0.4 58 ± 8
TetRi2-I59L L60M 8 ± 0.8 17 ± 0.9 3 ± 0.1
TetR i2 -D95A N81S 81 ± 2.7 2 ± 0.2 54 ± 4
TetRi2-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
Trang 4improved repression with 4-ddma-atc The same
exchange leads to one of the best 4-ddma-atc specific
reverse phenotypes in TetRi2-V99G Another very
spe-cific reverse phenotype is found for TetRi2-V99N with
a 34-fold better repression in the presence of
4-ddma-atc compared to 4-ddma-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 TetRi2 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
TetRi2variants with mutations at leucine 17 The effects of all possible exchanges of leucine at position 17 in the wild-type or TetRi2 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 TetRi2 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 TetRi2-L17M or -L17I exchanges show increased repression with atc, but not with 4-ddma-atc Exchanges leading to 4-ddma-atc dependent repression in TetRi2 include the aromatic amino acids W and Y, the hydroxyl containing resi-dues S and T, and C TetRi2-L17A is the best rev-TetRi2 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 TetRi2-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 TetRi2-V99G TetRi2-V99G is one of the best revTetRi2variants 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 TetRi2 -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 l M 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.
Trang 5analyzed 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 TetRi2
-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 TetRi2-L17A and TetRi2-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] TetRi2 -L17A was purified to homogeneity employing the protocol described for wild-type TetR [11] The purifi-cation protocol for TetRi2-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 MgCl2 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.
Trang 6[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 TetRi2 In contrast, 4-ddma-atc binding to
the revTetRi2 variants showed large cooperativity
Scatchard analysis confirmed positive cooperativity for
TetRi2-L17A and -V99N (Fig 5) The resulting
equi-librium binding constants are summarized in Table 2
As we could not saturate atc binding to TetRi2-L17A
and -V99N at 5 lm of atc, we assume constants below
2· 105 m)1 The equilibrium binding constant of atc
to TetRi2 was determined previously by Mg2+ -depend-ent titrations [5] and yielded a KA of 1.7· 107 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 revTetRi2 variants exhibit higher affinities for 4-ddma-atc compared to atc but the affinities are lower than the respective ones to TetRi2
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 l M and 0.005 l M [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 l M , 0.01 l M 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 MgCl2 concentrations [5] TetRMcorresponds to one monomer which can bind one [tc-Mg] + TetRDrepresents the dimer that binds [tc-Mg] + which can then bind the second molecule.
Equilibrium binding constants a , (·10 7
M )1)
TetR TetR i2
TetRi2 -V99N
TetRi2
TetRi2 -V99N
TetRi2
- L17A TetR M + [tc-Mg]+? TetR M [tc-Mg]+ 0.3b 132b 119600b 1.7b < 0.02c < 0.02c
TetRD[tc-Mg] + + [tc-Mg] + ? TetRD[tc-Mg] +
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.
Trang 7TetO binding in the presence of both effectors was
qualitatively analyzed by EMSA for the revTetRi2
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
TetRi2-V99N with 4-ddma-atc as corepressor, while
TetRi2-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 TetRi2 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 TetRi2 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 TetRi2-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 TetRi2 V99S has unaltered properties in the wild-type TetR, but leads to loss of DNA binding and 4-ddma-atc dependent corepression in TetRi2 V99T, on the other hand, shows partial DNA binding, corepression
by atc and induction by 4-ddma-atc when introduced
in TetRi2 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 TetRi2while 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 TetRi2-L17A and -V99N revealed reduced binding constants for 4-ddma-atc and 4-ddma-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 Coopcoop-erativity 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 l M )
car-rying tetO or a nonpalindromic sequence (usp DNA) were
incuba-ted for 15 min with 0.3 l M , 0.9 l M or 1.8 l M 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.
Trang 8L205S may be due to changes in the positioning of the
effector contacting residue H100 [7] This assumption
could apply to the properties of TetRi2-V99N, but the
exchanged residue in TetRi2-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 readreposition-ing 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 coliDH5a was used for cloning The DNA
con-taining randomized codons for helices a1 and a6 from
pWH1925 [8] were introduced in pWH1925-tetRi2
(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¢-aataagcgggccctactggatgcgctggcggt
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 tetRi2
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
37C 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 TetRi2-L17A to homogeneity was performed as described [11] For purifica-tion of TetRi2-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 MgCl2, 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 N2 after adjustment to 6 mm EDTA and thawed at 37C 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
Trang 94-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 MgCl2
0.1 lm, 0.01 lm or 0.005 lm atc or 4-ddma-atc were
titra-ted with TetR concentrations from 2· 10)10m to
1· 10)5m 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 94C 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 MgCl2 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 mm
MgCl2 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
Trang 10with 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