(Received for publication, January 31, 1996, and in revised form, December 13, 1996)
From the Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Federal Republic of Germany
A set of deletions and substitutions to alanine
was introduced into the loop separating helices 8 and
9 of
Tn10 Tet repressor (TetR). This region appears as an
unstructured loop in the crystal structure of the
TetR(D)·([Mg-tc]+)2 complex and is the only
internal segment of variable length in an alignment of Tet repressors
from seven different resistance determinants. In vivo
analysis of 10 mutants shows that this loop is important for
inducibility by tetracycline (tc), whereas DNA binding is not or only
marginally affected. All deletions have an induction-deficient
TetRS phenotype, but the corresponding substitutions do not
or only slightly affect inducibility. The purified mutant TetR proteins have a reduced affinity for tc in vitro that correlates
with their lack of inducibility. The association rate of
[Mg-tc]+ to the TetR mutants is enhanced. Since none of
the mutated residues contacts tc directly in the crystal structure, we
propose that the length of the loop is important for the structural
transition between a closed, tc binding and an open, operator binding
conformation of TetR. We propose that the deletions in the loop shift
the equilibrium between both forms toward the open, operator binding
conformation.
Binding of small effector molecules often triggers conformational changes in proteins. In the absence of bound ligand an open conformation is favored, whereas the presence of a ligand stabilizes the closed form (1). This is also the case for proteins involved in transcriptional regulation like PurR (2, 3), LacI (4), TrpR (5, 6), and the cAMP receptor protein (CRP)1 (7, 8). The active DNA binding conformation requires the bound ligand for PurR, TrpR, and CRP, whereas the ligand of LacI is an inducer leading to a conformation inactive in DNA binding. When bound, the ligand is mostly buried in a deep pocket in the protein interior and cannot reach or leave its binding site without inducing conformational changes. Thus, the liganded form corresponds to the closed conformation, and the unliganded form represents the open conformation.
Tet repressors (TetR) are well characterized isofunctional proteins
with at least two different conformations. They regulate the expression
of seven (classes A-E, G, and H) tetracycline (tc) resistance
determinants present in Gram-negative bacteria. In the absence of tc,
TetR is bound via an -helix-turn-
-helix motif (HTH) to
tetO. In the presence of tc, TetR dissociates rapidly from
tetO allowing expression of tc resistance (for a review, see
Ref. 9). Two crystal structures of the induced
TetR(D)·([Mg-tc]+)2 complex have recently
been solved (10, 11). The recognition helices of the TetR HTH are
separated by 39 Å and enclose an angle of about 110°. These helices
must approach each other by 5 Å and at least 50° to be able to bind
to B-form DNA (9). The inducer, [Mg-tc]+, is bound in a
deep pocket formed by residues from both monomers in the TetR dimer
(10, 11). Thus, the tc-bound form would correspond to the closed
conformation, and the DNA-binding form may represent the open
conformation.
Loop movements are often involved in conformational changes in proteins
where they contribute to the formation of ligand-binding sites and
enzymatically active structures. Owing to their flexibility, these
loops are not always defined in crystal structures (12). In TetR(D),
the loop from residues Ala-154 and Pro-167 separating helices 8 and
9 is flexible and leads only to weak electron density in the
TetR(D)·([Mg-tc]+)2 complex (11).
Comparison of the residues in this loop in an alignment of TetR
sequences (13) shows that it contains a region variable in both primary
structure and length (see Fig. 1 for a schematic representation of
TetR). Its length ranges from 11 residues in TetR(E) to 18 residues in
TetR(C) (11) and is the only internal segment of TetR that is variable
in length. Substitutions of single amino acids and deletions of 3 and
16 amino acids in this variable loop have shown that it is important for inducibility by tc but not for DNA binding (13, 14). Mutational analyses suggest that this region might be involved in the
conformational change associated with induction (13, 15). Residues in
this variable loop do not directly contact tc but might indirectly be
involved in tc binding by positioning of
9 (11).
We investigated the role of this variable loop in induction by introducing deletions and substitutions in TetR(B). Deletions led to induction deficiency of TetR(B), and the TetR variants bind tc with a reduced affinity. We suggest that these mutations act by interfering with the initial closure of the tc-binding pocket.
Chemicals were from Merck
(Darmstadt), Serva (Heidelberg), Sigma (München), or Roth
(Karlsruhe) and of the highest purity available. Tc was from Fluka
(Buchs). Enzymes for DNA restriction and modification were from
Boehringer Mannheim, Life Technologies, Inc. (Eggenstein), New England
BioLabs (Schwalbach), or Pharmacia (Freiburg). Isolation and
manipulation of DNA was as described (16). Sequencing was carried out
according to the protocol provided by Pharmacia for use with T7 DNA
polymerase, with [-32P]dATP from Amersham
(Braunschweig). Soluble protein extracts were prepared and analyzed in
Western blots as described (13).
All bacterial
strains are derived from Escherichia coli K12. Strain DH5
((17);
hsdR17(rK
mK+),
recA1, endA1, gyrA96, thi,
relA1, supE44,
80dlacZ
M15,
(lacZYA-argF)U169) was used for general
cloning procedures. Strains JM101 ((18);
(lac-proAB),
thi, supE, F
: traD36,
proAB, lacIqZ
M15) and
RZ1032 ((19); HfrKL16 PO/45, lysA(61-62), dut1,
ung1, thi1, relA1, supE44,
Zbd-279::Tn10) were used in the course of oligonucleotide-directed mutagenesis. Strain WH207 ((20);
lacX74, galK2, rpsL,
recA13) served as host strain for
-galactosidase assays.
Strain RB791 ((21); IN(rrnD-rrnE)1,
lacIqL8) was used for overexpression
of TetR variants. The plasmids pWH1200, pWH1201, pWH510 (22), pWH520
(23), and pWH1919 (14) that were used in the in vivo studies
have been described. The plasmids pWH1919 and pWH620 are derivatives of
pWH520. They differ from pWH520 only in the number of additional single
restriction sites in their tetR genes. PWH1919 contains
restriction sites for BstXI and MluI (14),
pWH620, in addition to the BstXI and MluI sites,
also sites for SauI and NcoI (see below). The
plasmid pWH1950 used for overexpression of TetR variants has also been described (24). The
phage tet50 (20, 25) used in the
-galactosidase assays and the M13mp19 derivative mWH892 (14) used
for oligonucleotide-directed mutagenesis have been described.
Mutations were introduced into the tetR gene by PCR according to the three-primer method of Landt et al. (26). The conditions for the first PCR were as follows: 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 100 µg/ml bovine serum albumin, 200 µM dNTP, 3 µM of the primer 1 and the mutagenic primer each, 636 pM pWH1919, 7.5 units of Pfu DNA polymerase (Stratagene, Heidelberg) in a total reaction volume of 150 µl; 25 cycles: 2 min/94 °C, 2 min/45 °C, 1 min 30 s/72 °C. One third (15 µl) of the purified (Promega Magic PCRpreps, Heidelberg) amplification products from the PCR-I was added to the reaction mixture of the PCR-II (buffer as in PCR-I, 400 µM dNTP, 2.5 µM primer 2, 795 pM pWH1919, 5 units of Pfu DNA polymerase in a total reaction volume of 100 µl; same program as in PCR-I). The 352-bp amplification products of the second PCR were purified, digested with NdeI and MluI, and cloned into likewise digested pWH1919 and pWH510. The entire 193-bp NdeI/MluI fragment was sequenced to ensure that only the desired mutation was present. For overexpression, the respective pWH1919 derivative was digested with XbaI and SphI. The resulting 711-bp fragment carrying the mutant tetR gene was ligated into likewise digested pWH1950.
Construction of Plasmids for the Constitutive Expression of tetR(E)A restriction site for NcoI was introduced 18 nucleotides downstream of the stop codon of the tetR(B) gene
located on mWH892-SauI by the method of Kunkel et
al. (19) and following the experimental procedures of Su and
El-Gewely (27). The presence of the mutation was verified by
restriction digestion and sequencing. The tetR(B) gene with
the novel restriction site was cloned as 711-bp
XbaI/SphI fragment from replicative form
mWH892-SauI/NcoI into likewise digested pWH520.
The resulting plasmid was named pWH620. The tetR(E) gene was
amplified by PCR (buffer as in PCR-I (see above), 17 nM
pWH905 (28), 1 µM each of the amplification primers
E-XbaI (5GATAGTG
TCTAGACTAAGCTTGGACGACG3
; the start
codon of tetR(E) is underlined and the XbaI site
is emphasized in boldface), and E-NcoI
(5
GACACATCCATGGCA
TTTATTACCATCC3
; the stop codon of tetR(E) is underlined and the NcoI site
is given in boldface), 200 µM dNTP, 1.25 units of
Pfu DNA polymerase; program see above). The resulting 657-bp
PCR product was digested with XbaI and NcoI and
ligated into likewise digested pWH620. The presence of
tetR(E) was verified by sequencing the entire gene. It was
cloned as 808-bp EcoRI/SphI fragment from
pWH620(E) into likewise digested pWH1200, yielding a pWH510 derivative
that was named pWH510(E).
Mutations in the HTH motif were
amplified by PCR (buffer as in PCR-I (see above), 200 µM
dNTP, 1 µM each of the flanking primers, 370 pM of the pWH1411 DNA carrying the respective mutation in the HTH motif (20, 29, 30), 6 units of Pfu DNA polymerase in
a total reaction volume of 150 µl; 1 cycle: 5 min/95 °C, 5 min/60 °C, 2 min/72 °C; 30 cycles: 2 min/94 °C, 2 min/60 °C, 2 min/72 °C). The 815-bp amplification products were
purified (Promega Magic PCRpreps), digested with XbaI and
SnaBI, and ligated into likewise digested pWH520 or
pWH520164-166. The 232-bp PCR product containing the HR44 mutation
was also ligated into likewise digested pWH1919NS82, pWH1919PT105, and
pWH1919DG178 (14). All PCR products cloned were completely sequenced to
ensure that no additional mutations had occurred.
DNA binding and inducibility by tc
was assayed in E. coli WH207(tet50). The phage
tet50 contains a tetA-lacZ transcriptional fusion (25) integrated in single copy into the WH207 genome (20). Cells
were grown in LB medium supplemented with the appropriate antibiotics.
-Galactosidase activities were determined as described by Miller
(31). Four independent cultures were assayed for each strain and
measurements repeated at least twice.
Derivatives of pWH1950
containing the tetR variants were transformed into
Escherichia coli RB791. Cells were grown in 1 liter of LB
medium at 37 °C in a 2-liter shaking flask, except for the strain
containing TetR(E) that was grown at 28 °C. The respective TetR
variant was overexpressed by adding
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 1 mM at an A600
0.9 and incubating the culture for an additional 4 h. Isolation
and purification of the protein was as described (24). Protein
concentrations were determined by the Bio-Rad protein assay (Bio-Rad,
München) and by saturating fluorescence titration with tc.
The concentration of tc
was determined by UV spectroscopy using an extinction coefficient of
355 = 13320 M
1
cm
1 in 0.1 N HCl (32). All fluorescence
measurements were carried out in a Spex Fluorolog equipped with double
Spex 1680 monochromators. The slit width was 2.2 mm for excitation (370 nm) and emission (515 nm), and an internal standard (8 mg/ml rhodamine
B (Kodak, Stuttgart) in 1,2-propanediol (Fluka)) was used to correct
for intensity fluctuations of the mercury lamp. Association constants K were determined under equilibrium conditions at 37 °C
as described (33) by fluorescence titration with limiting
Mg2+ concentrations. TetR monomer and tc were provided at
equimolar concentrations of 1 µM. Free Mg2+
concentrations ranging from 10
11 to 10
5
M were adjusted using diluted stock solutions of
MgCl2 and K buffer (100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM EDTA, and 5 mM
dithiothreitol) containing EDTA as a metal chelator (34). Free
Mg2+ concentrations ranging from 10
7 to
10
2 M were adjusted using diluted stock
solutions of MgCl2 and O buffer (100 mM
Tris-HCl, pH 8.0, 100 mM NaCl, and 5 mM
dithiothreitol) without EDTA. A KM of 2400 M
1 for the association of Mg2+ to
tc was employed to analyze the titration curves (35). The fitting was
performed by minimizing S2 =
(Fexp
Ftheor)2, where
Fexp and Ftheor are the
experimental and theoretical fluorescence intensities, respectively.
Fits with minimal S2 for a given pair of
K and were transformed to K for
= 1 (no cooperativity in tc binding) by the equation
K
= 1 =
0.5 × K
.2 Association
rate constants ka were determined at 20, 30, and
40 °C as described (32) with equimolar concentrations of TetR
monomer and tc at 25, 50, and 100 nM in F buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl2, and 5 mM dithiothreitol). All measurements were repeated at least twice.
We introduced the set of
mutations shown in Fig. 1 into the variable region of
TetR(B) to evaluate the importance of its length and sequence for DNA
binding and inducibility by tc. Deletion of the residues "DSM" in
TetR164-166 caused reduced in vivo inducibility (13). To
address the importance of these side chains in a "loss of contact"
experiment (36), they were changed to alanine (s164AAA). We also
mutated these three residues to "PTT" (s164PTT) to investigate the
effect of the junction (161-PTT
PPLL-170) created by the deletion without altering the length of the variable region. To determine the effect of the length of the variable region on TetR(B) activity, we deleted one (
162), two (
161-162), or three
(
161-163) residues in the segment between residues Thr-154-Asn-165
that is disordered in the
TetR(D)·([Mg-tc]+)2 crystal structure (10).
These residues were also substituted by alanine (TA162, s161AA,
s161AAA).
To determine the
in vivo operator binding activity of the tetR
mutants, they were transformed as pWH520 derivatives, which constitutively express TetR at a high level (37), into E. coli WH207(tet50). Repression of the
tetA-lacZ transcriptional fusion and inducibility by tc were
determined at 37 °C and are shown in the second and third columns of
Table I. All tetR variants show wt
operator-binding activity in that strain. Class E TetR on pWH620(E)
shows a marginally reduced inducibility with 86%
-galactosidase
activity compared with TetR(B). The same result was found for
TetRs161AAA. All other mutants with substitutions to alanine are fully
inducible. The substitution of residues 164-166 to "PTT" leads to
partial inducibility. In contrast, all deletions are less efficiently
inducible. Variants with deletions of three residues (
161-163,
164-166) are not inducible; the variant with a deletion of two
residues (
161-162) is only 2-fold and that with a one residue
deletion (
162) is 50-fold inducible.
|
The DNA-binding activity of all TetR variants and the inducibility of
the deletions of two and three residues are at the basal limit of
lacZ expression with 1% of the -galactosidase activity in unrepressed strains. The pWH520-based expression system provides no
resolution for large defects in inducibility (15) and slight differences in repression (29). To determine differences in the
DNA-binding activity and inducibility, we used a second expression system with a better resolution for mutants with wt-like binding activity (29). The mutants were recloned into pWH510 that expresses TetR at an at least 30-fold reduced level (23, 37). The repression mediated by wt TetR in WH207(
tet50) at 37 °C is only
14-fold in this construct, as compared with 100-fold in the
pWH520-background (Table I, column 2). Repression with the pWH510
derivatives is presented in column four of Table I. TetR
161-163,
TetR
161-162, and TetR
162 bind tetO with the same
efficiency as wt, the other mutations lead to a less than 2-fold
reduction in repression. Inducibility by tc was also determined and is
shown in column 5 of Table I. The increased sensitivity of the pWH510
derivatives reveals that the TetR variants are inducible to different
degrees. Only the deletions of three residues have a TetRS
phenotype. They are 2- (
164-166) and 7-fold (
161-163)
inducible. All other TetR variants tested are fully inducible at a
lower expression level.
The variable loop is clearly not important for DNA binding of TetR, since none of the mutations leads to significantly reduced repression of the tetA-lacZ transcriptional fusion. On the other hand, it is important for the inducibility by tc. Deletions of two and three amino acids lead to impaired inducibility, while none of the substitution mutants has a TetRS phenotype. We thus conclude that the deletions are mainly responsible for the TetRS phenotype. The slightly reduced inducibility of TetRs164PTT demonstrate that the amino acids at the positions deleted contribute to inducibility. Four effects can lead to a TetRS phenotype: (i) an increase in the protein steady-state level, (ii) a super-repressor, (iii) reduced binding of tc, and (iv) failure to release tet operator upon tc binding. Therefore, these questions were addressed.
Steady-state Levels of TetR MutantsThe steady-state levels
of the TetR variants encoded by pWH520 derivatives in E. coli WH207(tet50) were determined. Extracts of
soluble protein were prepared from log-phase cultures grown under the
same conditions as for the
-galactosidase activity measurements.
Aliquots of these extracts, which allow the detection of 2-fold changes
in TetR content (data not shown), were analyzed in Western blots for
their TetR content. A typical result is shown in Fig. 2.
The protein levels of the TetR mutants are either identical to the one
of wt, or only slightly reduced. This rules out (i) as a cause of the
TetRS phenotype and, in combination with the wt- or lower
DNA-binding activity of the mutants (Table I), also eliminates the
super-repressor explanation.
Binding of [Mg-tc]+ to TetR Variants
The TetR
variants of classes B and E, as well as the mutants s164PTT,
161-163, and
164-166 were overproduced and purified to
homogeneity. Their binding constants for [Mg-tc]+ were
determined under equilibrium binding conditions by fluorescence titration with limiting Mg2+ concentrations. The method
measures the increase in the intrinsic fluorescence of
[Mg-tc]+ upon binding of TetR and can be used to
determine binding constants between 1 × 1011 and
1 × 107 M
1 of tc
derivatives to TetR (35) and of tc to TetR mutants (15). The titration
curves obtained are shown in Fig. 3A. The
lowest free Mg2+ concentration needed for fluorescence
enhancement of tc is observed for TetR(E). Slightly higher
Mg2+ concentrations are needed for TetR(B) and TetRs164PTT,
in that order, while both TetR
164-166 and TetR
161-163 require
about 100-fold higher Mg2+ concentration to show
fluorescence enhancement. The best fits with the experimental data were
obtained with cooperativity values for
ranging from 0.5 (TetRs164PTT) to 18 (TetR(E)). Varying
between these values changed
the least square errors S2 (see "Experimental
Procedures") less than 5-fold (data not shown). Fig. 3B
shows the theoretical fluorescence calculated for TetR(B) at three
different cooperativity values of
. The three values for
chosen
were 0.5 (lowest value obtained for all mutants; dashed
line), 18 (highest value obtained for all mutants; full line) and 6, the value that lead to the lowest
S2 for TetR(B) (dotted line). The
experimental data fit all three curves reasonably well. This indicates
that the binding assay is not sensitive for potential cooperativity.
Since TetR(B) binds two molecules of tc without detectable
cooperativity as described (32), we determined the equilibrium
association constants K for
= 1. They are displayed in
the first column of Table II. The equilibrium
association constant determined for TetR(B) is with 1.6 × 109 very close to the previously published values of
1.9-3.1 × 109 (15, 32, 33) and that of TetR(E) is
about 2-fold higher. All mutants with a TetRS phenotype
have a reduced affinity for tc. While it is just 3-fold lower for the
substitution s164PTT, it is diminished about 70-fold for the deletions
of three amino acids (
161-163,
164-166). For all TetR(B)
mutants purified, the reduction in tc binding affinity correlates to
the severity of their in vivo TetRS
phenotypes.
|
A decrease in the equilibrium binding constant of tc to TetR can be
caused by a decrease in the association rate constant, an increase in
the dissociation rate constant, or both. The time-dependent association of tc to TetR can be fitted using second-order kinetics for
a bimolecular reaction (32). The association rates of tc to the TetR
variants were determined by measuring the increase in tc fluorescence
upon addition of Tet repressor at temperatures of 20, 30, and 40 °C.
They are shown in Fig. 4 and for ka at 30 °C in column three of Table II. TetR(B) has the lowest
association rate constant of all variants at all temperatures assayed.
At 30 °C, ka of tc to TetR(B) is 1.4-fold slower
than to TetR(B)161-163, 3.4-fold than to TetR(B)s164PTT, 5.2-fold
than to TetR(B)
164-166, and 11-fold than to TetR(E). The
temperature dependence is smallest for TetR(E), almost identical for
TetR(B) and the mutants
161-163 and s164PTT, and largest for the
mutant
164-166. The activation energies EA
for this reaction were calculated from the association rate constants
in Arrhenius plots and are displayed in column four of Table II. They
are the same as TetR(B) for TetRs164PTT and TetR
161-163, about
twice as high for TetR
164-166, but only half as high for TetR(E).
Thus, the reduction in tc binding affinity is not due to an impaired
access of the drug to its binding site, but tc must be retained less
efficiently in its binding pocket. We can imagine that the reduction is
caused either by the loss of direct contacts to tc or by interfering
with the conformational change that leads to the closure of the tc
binding pocket.
Combinations of TetRS Mutations with HTH Mutations
If the deletion mutants interfere with the
conformational change leading to the induced structure of TetR, the
conformational equilibrium between the DNA-bound and induced structures
of TetR will be shifted toward the DNA-bound form. This assumption
would imply that more DNA-binding active repressor is present in the mutants than in the wild-type and should lead to enhanced repression in
the in vivo test system. Screening for second-site
suppressors of the TetRS phenotype of TetR164-166
yielded one candidate with a HR44
mutation.3 The in vivo
repression conveyed by the double mutant HR44
164-166 was 2-fold
higher than that of the single mutant HR44 (Table III), even though the mutant with residues 164-166 deleted does not show
increased DNA-binding activity in vivo (Table I). This would be consistent with the idea of a shift in the equilibrium toward the
DNA-bound form. To determine whether the enhanced DNA-binding activity
observed in this case is specific for these two mutations or general
for combinations of a mutation in the DNA-binding domain with the
deletion
164-166, we combined the deletion mutant
164-166 with
10 other mutations in the DNA-reading head. They are located in both
-helices of the HTH (
2: TQ27, LR34, LW34;
3: QG38, TG40, LT41,
WG43), the connecting turn (VS36, VW36), and the following
-helix 4 (KT48) at positions located either in the protein interior or at the
surface (11). These HTH mutations span a wide range of DNA-binding
activities, with HR44 being at an intermediate level (20, 29, 30).
|
To additionally ask if mutations with a TetRS phenotype
generally enhance the DNA-binding activity of mutations in the HTH, we
combined the HR44 mutation as a control with three previously described
TetRS mutants that have phenotypes similar to 164-166
(15) and either contact tc directly (NS82, PT105) or do not form
contacts with tc (DG178).
The pWH520 derivatives
carrying the single and double mutants were transformed into
WH207(tet50) and their
-galactosidase activities
determined at 37 °C. The results are shown in Table III. While the
-galactosidase activities determined for the mutants with a
TetRS phenotype are identical to that of wt TetR, those of
the mutations in the HTH are between 3- and 60-fold higher. In
combination with the
164-166 mutation, the
-galactosidase
activities obtained for 9 out of the 11 HTH mutants are 1.5-3-fold
lower when compared with the respective single mutation. Only the two
mutations at position 34 with their unchanged
-galactosidase
activities do not show an increase in repression. Repression is reduced
in two of the double mutants involving HR44 (NS82, PT105), while it is increased in the other two mutants (
164-166, DG178).
Soluble protein extracts were prepared from log phase
cultures of pWH520 derivatives encoding the TetR variants in E. coli WH207(tet50) and analyzed in Western blots for
their TetR content. A typical result is shown in Fig. 5.
The intracellular protein levels of the two mutants with combinations
of a substitution at position 34 and
164-166 are strongly reduced.
For the other single and double mutants, the intracellular protein
levels determined are either identical to the wild-type or only
slightly reduced, as judged by their band intensities.
When only the double mutants with roughly unchanged protein levels are
considered, the increase in the DNA-binding activity of the HTH
mutations after introduction of the 164-166 deletion is general and
not specific for a certain mutation in the HTH. This effect is not
general for mutants with a TetRS phenotype, as the data for
the double mutants with NS82 and PT105 show.
Mutations in the variable region of TetR affect DNA-binding activity only marginally (Table I) verifying that they do not introduce major changes into the DNA-binding conformation of TetR. Deletions of two or three residues have an induction-deficient TetRS phenotype, and the single residue deletion is slightly impaired in inducibility. All deletion mutants show a clear correlation between the reduction in loop length and the severity of the TetRS phenotype. In contrast, the alanine substitution mutants corresponding to the deletions do not affect inducibility and one further substitution mutant (s164PTT) has only a small effect on induction (Table I). This demonstrates that the location of the deletion and the sequences of the deleted amino acids contribute less to the TetRS phenotype than the reduced length of the variable region.
What leads to the TetRS phenotype of the deletion mutants?
The roughly unchanged steady-state levels of the proteins indicate that
the TetRS phenotype is an intrinsic property of the TetR
variants. All TetRS mutants purified have reduced
affinities for tc (Table II) that correlate to the severity of their
in vivo induction deficiency (Table I). Therefore, the
deletions must change the tc binding pocket. Since none of the
mutations affects residues in direct contact to tc, the deletions
should indirectly exert their effects on tc binding. The deletion
length dependence suggests that positioning of residues within or
adjacent to the variable loop may be important for induction. The loop
might be indirectly involved in tc binding by positioning 9, so that
residues in
9 can form contacts with the D ring of tc (11). The end
points of helices
8 and
9 are separated by 24.3 Å in the crystal
structure of the TetR(D)·([Mg-tc]+)2
complex (10). A fully extended peptide chain in a
-sheet spans 3.6 Å per residue (38). If we assume that each residue in the loop only
spans 2.8 Å, 9 of the 14 residues present would suffice to bridge 25 Å. All deletion mutants would suffice for that. The end points of the
segment disordered in the crystal structure are separated by 19.5 Å.
This distance could be bridged by only seven residues, whereas nine are
present in the two-residue deletion mutant. Thus, if the residues were
fully extended they would not alter positioning of
9.
Substitutions at positions 7, 8, or 9 of tc that contact amino acids in
9 affect TetR binding and induction less than 10-fold (35, 39).
Twelve induction-deficient TetR mutants were isolated in
9.
Müller et al. (15) proposed that they affect
inducibility by preventing the structural change associated with
induction since, for all mutants, the TetRS phenotypes
observed in vivo are more severe than indicated by the
merely 1.3-4.5-fold reduced tc binding (see Tables I and IV in Ref.
15). Taken together, the loop deletions may affect tc binding by
interfering with the formation of contacts between
9 and tc or by
interfering with the conformational changes necessary for induction, or
both.
It has been suggested that the lack of induction of several single
TetRS mutants is not due to their reduced inducer binding
but to preventing conformational changes (15). Müller et
al. (15) proposed a tc-induced reorientation of the four-helix
bundle formed by 8 and
10 from both monomers. In this model,
9
serves as a "bolt" locking the
TetR(D)·([Mg-tc]+)2 complex in the induced,
closed structure. Shortening the variable region by more than one amino
acid might thus interfere with the closure of the tc-binding pocket
inhibiting the transition between the DNA-binding and the induced
conformations. TetR exists most likely in an equilibrium between the
DNA-binding and the induced structures. Mutations stabilizing the
DNA-binding conformation would be expected to show a TetRS
phenotype, increased in vivo repression in the absence of
inducer, and an increased association rate of tc. These are exactly the properties we observed for the deletion mutants. They show a clear TetRS phenotype. Owing to the law of mass action, the
induction deficiency is more pronounced in the high level expression
system (pWH520) than in the low level expression system (pWH510). The
effect on DNA binding is not detected for the deletion mutants alone
due to limitations of resolution in the in vivo measurements
but is clearly seen in combinations with additional mutations in the HTH having themselves reduced affinity to tetO. The
increased DNA binding is not caused by increased protein levels. The
facilitated access of tc to the binding pocket is indicated by the
increased association rates for [Mg-tc]+. It is slowest
with TetR(B) and faster in all mutants (Table II). Thus, the reduced
affinity for tc is not caused by preventing the drug from reaching its
binding site in the protein interior but must be due to its increased
dissociation from the binding pocket. Mutants of groups I and II
periplasmic sugar binding proteins show similar effects. A decrease in
both association and dissociation rate constants for the respective
ligand was observed and interpreted as an increased rigidity of the
closed conformation, thereby shifting the equilibrium between closed
and open forms of the protein toward the closed form. This assumption
was confirmed by structural analysis (40, 41). It therefore seems
plausible to assume for the TetR mutants that shortening the variable
loop interferes with the closure of the tc-binding pocket, thus
shifting the equilibrium between the DNA- and tc-binding conformations
toward the open DNA-binding conformation.
No correlation was seen between the activation energies of tc association and the in vivo phenotype of the TetR variants. Further experiments are needed to determine why the activation energies for tc association vary among different inducible and non-inducible TetR variants.
Taken together, we suggest that the deletions in the variable region of TetR affect the closure of the tc-binding pocket upon association of tc. This occurs most likely by interfering with the conformational change that locks the TetR(D)·([Mg-tc]+)2 complex into the induced structure.
We thank K. Pfleiderer for constructing pWH620 and H. Prell for typing the manuscript.