Tet repressor residues indirectly recognizing anhydrotetracyclinePeter Schubert*, Klaus Pfleiderer and Wolfgang Hillen Lehrstuhl fu¨r Mikrobiologie, Institut fu¨r Mikrobiologie, Biochemi
Trang 1Tet repressor residues indirectly recognizing anhydrotetracycline
Peter Schubert*, Klaus Pfleiderer and Wolfgang Hillen
Lehrstuhl fu¨r Mikrobiologie, Institut fu¨r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universita¨t Erlangen, Germany
Two tetracycline repressor (TetR) sequence variants sharing
63% identical amino acids were investigated in terms of their
recognition specificity for tetracycline and
anhydrotetra-cycline Thermodynamic complexstabilities determined by
urea-dependent unfolding reveal that tetracycline stabilizes
both variants to a similar extent but that anhydrotetracycline
discriminates between them significantly Isofunctional
TetR hybrid proteins of these sequence variants were
constructed and their denaturation profiles identified
resi-dues 57 and 61 as the complexstability determinant
Association kinetics reveal different recognition of these
TetR variants by anhydrotetracycline, but the binding
constants indicate similar stabilization The identified
residues connect to an internal water network, which suggests that the discrepancy in the observed thermo-dynamics may be caused by an entropy effect Exchange of these interacting residues between the two TetR variants appears to influence the flexibility of this water organiza-tion, demonstrating the importance of buried, structural water molecules for ligand recognition and protein func-tion Therefore, this structural module seems to be a key requisite for the plasticity of the multiple ligand binding protein TetR
Keywords: Tet repressor; gene regulation; protein stability; ligand binding; antibiotic resistance
The biological function of many proteins is triggered and
modulated by binding of effector molecules or a variety of
extrinsic cofactors that greatly expand the repertoire of
cellular processes executed by polypeptides, DNA or small
proteins [1] Therefore, molecular recognition is a
funda-mental process in all living systems, regulating processes as
diverse as transcription, cell signalling and immunity [2–4]
Recognition mechanisms may be divided into two general
categories, named specific and related specificities [5] For
example, mature immunoglobulins (Ig) are highly specific
while those in the germline bind a broad range of antigens
[6] This effect is explained by a diversity of conformations
for the related specificity germline antibody, a pre-existing
subpopulation of antibody isomers based on increased
flexibility [7,8]
To understand the thermodynamic and kinetic principles
of protein ligand binding in more detail, concepts of energy
landscapes and folding funnels were used [9]
Characteriza-tion of binding sites revealed that such regions are usually
depressions in the protein surface where a greater average
degree of exposure of hydrophobicity groups occurs [10]
However, for a detailed knowledge of protein specificity at
the molecular level it is essential to understand the mechanisms of protein–ligand recognition by obtaining information about the structure, energetics and dynamics of the free and complexed species under a variety of condi-tions We used two sequence variants of the tetracycline repressor (TetR) to investigate ligand recognition of two different tetracycline (tc) derivatives, tc and anhydrotetra-cycline (atc) TetR regulates resistance to the antibiotic tc in Gram-negative bacteria by inducer binding [11] This system
is successfully adapted for regulation of gene expression in different organisms [12] Based on sequence similarities of isolates from various bacteria TetR variants were grouped into nine classes called A to E, G, H, J and 30 [13] The proteins share between 38 and 88% sequence identity and are presumably isostructural and isofunctional Each homodimeric protein consistes of an N-terminal DNA-binding domain connected to a core domain harbouring the dimerization motif and the effector binding site Repression
of gene expression occurs by specific binding to tetO via a helix–turn–helix motif Binding of the effector molecule leads to a conformational change resulting in the loss of DNA binding and initiation of transcription [11,14] The crystal structure of TetR(D)[tc–Mg]2indicates the position-ing of the inducer inside the protein core and reveals interactions of the drug with both monomers [15,16] Here we studied the thermodynamic complexstability and binding affinity of the two naturally occuring TetR variants B and D with tc and atc The sequence identity between these two TetR variants is only 63%, but it includes most residues involved in operator and DNA binding The sequence identity for the helix–turn–helix domain is 94% and for the residues contacting tc it is 68% Urea-dependent unfolding yields similar stabilization of the two sequence variants by tc binding However, atc shows a strong discrimination between TetR(B) and TetR(D) in terms of stabilization Using TetR(B/D) hybrid proteins we have
Correspondence to W Hillen, Lehrstuhl fu¨r Mikrobiologie, Institut fu¨r
Mikrobiologie, Biochemie und Genetik,
Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Staudtstraße 5, 91058 Erlangen,
Germany Fax: + 49 91318528082, Tel.: + 49 91318528081,
E-mail: whillen@biologie.uni-erlangen.de
Abbreviations: TetR, tetracycline repressor; tc, tetracycline;
atc, anhydrotetracycline.
*Present address: Biomedical Research Centre, University of British
Columbia, Vancouver, V6T 1Z3, Canada.
(Received 5 February 2004, revised 21 March 2004,
accepted 30 March 2004)
Trang 2narrowed the determinants for the different atc complex
stabilities to two residues localized at one side of the
tc-binding pocket, where they are involved in the last step of
the proposed induction mechanism of TetR by stabilizing
the induced complexby a water zipper [14]
Thermo-dynamic and kinetic investigations reveal that replacement
of these residues in TetR(D) to the ones found in TetR(B)
reduces complexstability and recognition of atc This
indicates that this water network is important for stability
and drug affinity, but not for induction
Experimental procedures
Material and general methods
Anhydrotetracycline (atc) was purchased from Acros (Geel,
Belgium), all other chemicals were from Merck (Darmstadt,
Germany), Roth (Karlsruhe, Germany) or Sigma
(Mu¨n-chen, Germany) at the highest available purity Enzymes for
DNA restriction and modification were from New England
Biolabs (Schwalbach, Germany), Boehringer (Mannheim,
Germany), Stratagene (Heidelberg, Germany) or
Pharma-cia (Freiburg, Germany) Oligonucleotides were from PE
Applied Biosystems (Weiterstadt, Germany) Sequencing
was carried out according to the protocol provided by
Perkin Elmer for cycle sequencing and sequence waas
analysed with an ABI PRISMTM310 Genetic Analyzer (PE
Applied Biosystems, Weiterstadt, Germany)
Bacterial strains and plasmids
All bacterial strains were derived from Escherichia coli K12
Strain DH5a [hsdR17(rKmK+), recA1, endA1, gyrA96, thi,
relA1, supE44, /80 dLacZ(M15, [(lacZYA-argF)U169] was
used for general transformation procedures Strain WH207
(lacX74, gaK2, rpsL, recA13) [17] served as host strain
for b-galactosidase assays The plasmids pWH806 and
pWH853(B) [17] and pWH853(D) [18] used in the in vivo
assay as well as pWH1950 [19] for overexpression have been
described before
Construction of the chimeric tetR genes
All tetR variants were constructed by PCR according to the
three-primer method [20] The products of the second PCR
reaction were purified and digested with XbaI/MluI or
MluI/NcoI and cloned in pWH853 to replace the respective
position of tetR For overexpression these constructs were
digested with XbaI and NcoI and cloned into likewise
digested pWH1950 DNA of positive candidates was
analysed by sequencing of tetR
b-Galactosidase assay
Repression and induction efficiencies of the TetR variants
were assayed in E coli WH207ktet50 carrying the respective
pWH853 derivatives The phage ktet50 contains a tetA–
lacZtranscriptional fusion [17] integrated as single copy into
the WH207 genome Bacteria were grown at 28C in
Luria–Bertani medium supplemented with the appropriate
antibiotics Quantification of induction efficiencies was
carried out with 0.2 lgÆmL)1atc in overnight and log phase
cultures b-Galactosidase activities were determined as described by Miller [21] Three independent cultures were assayed for each strain and measurements were repeated at least twice
Purification of the TetR variants pWH1950 derivatives of the different constructs were transformed into E coli RB791 Cells were grown in 3 L
of Lurian–Bertani medium at 28C in shaking flasks TetR expression was induced by adding isopropyl thio-b-D -galactoside to a final concentration of 1 mM at D600 ¼ 0.7–1.0 Cells were pelleted and resuspended in buffer A containing 50 mM NaCl, 2 mM dithiothreitol, 20 mM sodium phosphate buffer pH 6.8 and broken by sonication TetR variants were purfied by cation exchange chromato-graphy and gel filtration as described [19] The amounts of the proteins were obtained from the UV absorption at
280 nm [22] and their activities were determined by titration with atc [23]
Fluorescence and CD spectroscopy Fluorescence intensities were measured with a SpexFluo-rolog 1680 double spectrometer in 1 cm cells at protein concentrations of 1 lMor 5 lM Excitation was at 280 nm and emission was recorded at the maximum of the difference between the native and the denatured fluores-cence spectrum The bandwidth for excitation and emission was 2.2 mm CD measurements were carried out on a Jasco J-715 spectropolarimeter in 0.5 cm cells at protein concen-trations of 5 lM TetR monomer The TetR[atc–Mg]2 complexwas formed by adding 10 lMatc
Unfolding of the TetR complexes, thermodynamic and kinetic constants
We used F-buffer containing 100 mMNaCl, 100 mMTris/ HCl pH 7.5, 5 mMMgCl2, 1 mMEDTA, 1 mM dithiothre-itol for all spectroscopic measurements Urea was obtained from ICN Biochemicals (Eschwege, Germany) and urea solutions were prepared each day Equilibrium denaturation was performed by incubating protein samples overnight at the indicated urea concentration Renaturation reactions were achieved by incubating the samples overnight at 8M urea and then diluting them 200-fold with F-buffer All reactions were performed at 22C and all TetR concentra-tions relate to the monomer Tetracycline or its derivative (xtc) was used in a onefold molar excess over protein Thermodynamic calculations of the urea-induced denatur-ation of the TetR[tc/atc–Mg] complexvariants were performed as described before [24,25] by extending the calculation as it applies to the monomeric (¼ TetR) as follows:
2TetRNþ 2xtc þ 2Mg2þ¼ ½TetRxtcMg2
¼ 2TetRUþ 2xtc þ 2Mg2þ where N is native and U is unfolded
The left side of the equation shows the association/ dissociation equilibrium and the right side the folding/
Trang 3unfolding equilibrium For the unfolding process the
equilibium constant (KU) could be given as:
KUỬ ơU2ơMg2ợ2ơxtc2=ơTetRxtcMg2
The equilibrium constant for ligand-free systems is given as:
KUỬ 2Ptf2U=fNơ24
where Ptis the total protein concentration
Mass balance yields:
ơMg2ợtỬ ơMg2ợ ợ ơCN
ơxtctỬ ơxtc ợ ơCN
where [CN] is the concentration of the native complex, resulting in:
KUỬ 2PtfUđơMg2ợt PtfNỡ2đơxtct PtfNỡ2=fN where fN and fU are the fraction native or unfolded, respectively This equation was used to calculateDG in the different states of the unfolding pathway
Mg2+-independent and -dependent atc equilibrium association constants were determined as published [26] The association rate constants were determined at 28C [27] with equimolar concentrations of TetR monomer and atc in F-buffer as mentioned above All experiments were repeated at least twice
Fig 1 TetR crystal structure and amino acid composition (A) Structure of TetR[tcỜMg] 2 Monomers are shown in blue and red, helices are indicated by numbers in the blue monomer and Tc is shown in green (B) Alignment of TetR(B) and TetR(D) Conserved residues are shown in reverse type Tc binding residues (black filled point) and residues involved in coordinating the water zipper (Ỉ) are indicated [14,30].
Trang 4Unfolding of TetR complexed with tc or atc
We used urea-dependent denaturation as described before
[24,25] to determine the stabilities of TetR[atc–Mg]2
com-plexes in comparison with TetR[tc–Mg]2 As shown by the
crystal structure of the latter complexbinding of tc occurs
inside the TetR dimer (Fig 1A) [15] The two TetR B and D
sequence variants share 63% amino acid identity (Fig 1B)
and fold into the same quaternary structure in the free and
tc-complexed forms [15] Structural changes during
unfold-ing of the complexes were observed by fluorescence and CD
As the fluorescence and CD properties of the two proteins in
their complexed forms are analogous, we show only the
data for the TetR(D)[atc–Mg]2 complex The main
fluor-escence of the TetR[atc–Mg]2complexoriginates from atc
and four naturally occuring trp residues The emission
spectrum excited at 280 nm shows two maxima at 362 nm
and 515 nm (Fig 2A), resulting from trp emission [22]
and energy transfer from trp to atc [28], respectively In the
denatured state at 8Murea the trp emission maximum shifts
to at 354 nm and is slightly increased in intensity, but the
515-nm band is absent, indicating the loss of ligand binding
(Fig 2A) Therefore, the intensity change of the 515-nm
band was used to follow denaturation The TetR[atc–Mg]2
complex ex cited at 455 nm shows also an emission max
i-mum at 515 nm which is also apparently absent at 8Murea
(Fig 2B) This fluorescence change of atc was also used to
monitor complexdenaturation to compare with energy
transfer For the respective tc-complexes unfolding was
followed by the change of the energy transfer band at
508 nm and the tc fluorescence at 508 nm excited at 370 nm
(data not shown)
The TetR[atc–Mg]2 complexshows CD minima at
208 nm and 222 nm (data not shown) reflecting the high
content of a-helical structure [15] The CD spectra in the
presence or absence of the inducer are the same in that curve
segment Since both absorption bands are absent at 8M
urea the ellipticity at 222 nm was also used to quantify
TetR[atc–Mg]2unfolding Each of the three probes yields
identical results when used to observe denaturation,
show-ing monophasic, sigmoidal curves indicatshow-ing the absence of
stable folding intermediates (Fig 2C) This demonstrates
that denaturation of the protein fold as observed by CD,
and release of the ligand as observed by fluorescence, occur
simultaneously As expected for a bimolecular reaction, the
midpoint of the unfolding transition depends on the protein
concentration (Table 1) The transition midpoints shift
from 6.2Mto about 6.5M, when the protein concentration
is increased fivefold To analyse the efficiency of the
refolding reaction the TetR(D)[atc–Mg]2 complexwas
treated with 8M urea and subsequently diluted 200-fold
with urea-free buffer The fluorescence emission spectrum
and CD of these renatured complexes were identical with
those obtained from the native form (Fig 2A,B) We
conclude that denaturation under these conditions is a
completely reversible, single-step reaction for the TetR[atc–
Mg]2 and the TetR[tc–Mg]2complexes This allows
quan-tification using the two-state model [29] in which only folded
complexes, unfolded monomers and free ligand exist at
equilibrium in significant concentrations
Thermodynamic stabilities of TetR[tc–Mg]2and TetR[atc–Mg]2complexes
Extrapolation of the urea-induced unfolding curves to 0M urea (Fig 2C, inset) to calculate the Gibbs free energy of unfoldingDGU(H2O) gave the results shown in Table 1 The DG (HO) of 123 kJÆmol)1 for TetR(D)[atc–Mg]
Fig 2 Fluorescence probes to determine complex stability (A) Fluor-escence emission spectra of TetR(D)[atc–Mg] 2 ; native (top curve), re-natured (dotted line below) and dere-natured (dashed line) (B) Emission spectra of atc; native (top curve), renatured (dotted line, below) and denatured (dashed line) (C) Urea-induced unfolding curve of TetR(-D)[atc–Mg] 2 Unfolding was followed by different probes (s, energy transfer; d, atc fluorescence; m, CD) and extrapolated for determin-ation of complexstability (inset).
Trang 5represents a stabilization of 69 kJÆmol)1compared to the
free protein [25] In contrast, the stabilization of TetR(B) by
complexformation with [atc–Mg]+ is only 26 kJÆmol)1
Although free TetR(D) is less stable than TetR(B) [25] it is
stabilized more by binding of [atc–Mg]+ The complexes of
the two sequence variants with [tc–Mg]+are stabilized by
13 kJÆmol)1 and 12 kJÆmol)1, respectively This indicates
that the TetR(D) sequence variant must undergo more
favourable interactions with atc than does TetR(B), despite
of the fact that 63% of the amino acids (Fig 1B) and
the folds of the polypeptide chains are identical These
interactions must also be specific for atc as no difference is
observed for the tc complexes
Interaction of TetR(B/D) chimera with [atc–Mg]+
We used some TetR(B/D) hybrid proteins from previous
work [18,25] and constructed five new chimeras based on
the assumption that residues contacting tc in the
TetR(D)[tc–Mg]2 structure (Fig 3) should be involved in
binding of atc as well Residues contacting [tc–Mg]+are
marked with open circles in Fig 1B The chimeric proteins
analysed in this work for binding and affinity to [tc–Mg]+
and [atc–Mg]+are shown in Fig 4
To determine both their in vivo repression efficiency and
inducibility all chimeric genes were cloned into pWH853
[17] and transformed into E coli WH207ktet50 containing a
chromosomal tetA–lacZ-fusion The b-galactosidase activ-ities at 28C were determined in the presence and absence
of 0.2 lgÆmL)1 atc As shown in Table 2 none of the mutants shows a significantly reduced inducibility com-pared to TetR(D) The repression efficiencies are nearly identical to TetR(B), only TetR(D) is a less efficient repressor and is also less inducible as observed before [18] The TetR(B/D) chimeras were overexpressed and purified
to homogenity as described previously [19] All purified TetR variants show similar spectral properties in the free and complexed forms (data not shown) The unfolding of the free and complexed proteins was carried out by urea-induced denaturation as mentioned above showing identical denaturation pathways (data not shown) indicated by monophasic denaturation curves The thermodynamic stabilities given as transition midpoints are summarized in Table 2 Tc binding results in a similar stabilization for all sequence variants For atc only TetR(B/D)51–208 exhibits TetR(D)[atc–Mg]2-like stability, all other chimera show TetR(B)[atc–Mg]2-like stability These data indicate that residues between positions 51 and 63 are responsible for the differences in [atc–Mg]+binding
Identifying single residues for atc recognition Three residues of the segment 51–62 are different between TetR(B) and TetR(D) (Fig 1B) The mutants TetR(B)57/
Table 1 Thermodynamic stability of the TetR(B)[tc/atc–Mg] 2 and TetR(D)[tc/atc–Mg] 2 complexes.
DG U
[kJÆmol)1]
Urea 1/2
[ M ]
DG U
[kJÆmol)1]
Urea 1/2
[ M ]
DG U
[kJÆmol)1]
Urea 1/2
[ M ]
a Urea-dependent unfolding was followed by the change of the energy transfer signal at the wavelength with the maximal difference of fluorescence between the native and the denatured forms, at protein concentrations of 1 l M (excitation at 280 nm) and 5 l M (excitation at
295 nm); tc and atc in onefold excess at 28 C b
The change of CD was observed at 222 nm.
Fig 3 TetR–tc interactions Binding to the different monomers is shown by blue and green symbols, respectively, and involved water molecules are depicted as red spheres For comparison the chemical structure of the atc molecule is shown to the right The two molecules have similar chemical structures, differing only in that the hydroxyl group at position 6 in tc and the neighbouring hydro-gen bond are eliminated in atc, resulting in an aromatic ring C.
Trang 659/61D and TetR(D)57/59/61B contain replacements of
these three amino acids by the respective other residues The
denaturation results shown in Table 2 demonstrate that
these three residues determine the stability difference of the
TetR–inducer complexes When they originate from the
TetR(B) sequence the atc-mediated stabilization is small,
whereas the TetR(D) residues lead to higher stabilization,
hence better recognition We then constructed all possible
double and single TetR variants for these positions in the
TetR(D) sequence background They show the same in vivo
repression and inducibility as TetR(D) (Table 2) The
denaturation data reveal that complexstability is strongly
affected when the residue at position 57 is exchanged, but
the alterations at positions 57 and 61 are necessary to yield
the fully reduced TetR(B)[atc–Mg]2-like stability Thus, they
are the determinant for the improved recognition of atc by
TetR(D)
Association rate constants for inducer binding
to the TetR(D/B) variants
From the thermodynamic point of view the complex
stability should reflect the equilibrium binding constant
(KA) of atc to TetR Therefore we determined KA for
TetR(B), TetR(D) and TetR(D/B) single, double and triple
mutants using an improved assay [26] which takes into
account the effect that atc binds Mg2+ independently of
TetR to a small extent The Mg2+ independent atc equilibrium constants (KT) of the TetR(D/B) constructs were determined by titration with inducer in the presence of EDTA following complexformation by atc fluorescence emission at 545 nm excited at 455 nm The KTvalues were calculated from these titration curves as described [26] and are listed in Table 3 The Mg2+-dependent binding constants were obtained from titrations of TetR complexed with atc in the presence of Mg2+ [26] The resulting association constants KA are also listed in Table 3 The value for TetR(D) is identical to that published before [26] The single exchanges of the residues at positions 57 and 61 show a partial alteration of affinity from the TetR(D) to the TetR(B) sequence variant, whereas the double mutation shows the full reduction Single and combined alterations involving position 59 show either no or smaller effects
Association rate constant of TetR[atc–Mg]2complex formation
The different equilibrium binding constants of atc for both TetR sequence variants could be caused by different association or dissociation rate constants, or both The time-dependent association rate constants of atc for both sequence variants were determined by measuring the increase in the atc fluorescence upon addition of TetR as described [27] They were fitted using second-order kinetics for a bimolecular reaction and the results are also presented
in Table 3 showing a kass value of 1· 10)6M )1Æs)1 for TetR(B) and a sevenfold higher rate constant for TetR(D) Since this accounts for the total difference seen in equilib-rium constants the different stability of TetR[atc–Mg]2 is based only on different association rates and therefore on molecular recognition
Discussion
In this study we used urea-induced unfolding to determine the thermodynamic stabilities of two TetR sequence vari-ants B and D complexed with the inducer atc or tc The change of the spectral probes used show identical results reflecting the coordinated destruction of the tertiary struc-ture, the loss of the ligand binding and the break down of the secondary structure The monophasic, sigmoidal curves for urea-dependent unfolding allow the use of a two-state model for calculating thermodynamic complexstabilities The values for the Gibbs free energyDGU(H2O) for the two TetR sequence variants B and D complexed with tc were determined to 83 kJÆmol)1 and 66 kJÆmol)1, which com-pared to their free forms [25] reveals that tc stabilizes both sequence variants to similar extents of 13 kJÆmol)1 and
12 kJÆmol)1, respectively However, atc binding results in complexstabilities of 96 kJÆmol)1 for the B and 123 kJÆ mol)1 for the D variant, which leads to stabilizations of
26 kJÆmol)1and 69 kJÆmol)1, respectively
This surprising stabilization difference of atc with the TetR variants B and D is reflected in the respective association constants The increased affinity for TetR(D) compared to TetR(B) is accounted for by different associ-ation rate constants, thus indicating the identical overall structures of TetR(D)[tc–Mg]2 and TetR(B)[tc–Mg]2 [15] that is used to identify the determinant for this difference in
Fig 4 Overview of the chimeric TetR(B/D) constructs The respective
wild-type proteins are shown by filled (TetR(D)) and open (TetR(B))
bars The hybrid proteins are shown with their designation given on
the right The top panel indicates the location of the TetR residues
interacting with tc (Fig 1B).
Trang 7ligand stabilization The sequence alignment of B and D
shows that the 16 residues that interact with the inducer
(Fig 3) are highly conserved (Fig 1B) The fact that both
sequence variants form identical primary contacts to the
inducer supports the hypothesis that the different affinities
must be due to an indirect effect Functional TetR(B/D)
hybrid proteins enabled us to narrow the determinant for
the different stabilities to the residues 57 and 61, located at
the C terminus of the hinge helix a4 connecting the DNA
binding domain with the protein core (Fig 5) Although
located close to residue His64 making contact to tc in the
crystal structure [15], these residues are too far away to
exert a direct influence The replacement of the
solvent-exposed Val by the chemically similar Ile residue at position
57 should cause just a small effect, but this exchange
contributes the most to destabilization This may be explained by a special feature observed in the crystal structure of the TetR(D)–tetO complex[14]
The comparison of the crystal structures in the tc-induced with the DNA-bound forms leads to a proposed induction mechanism [14] After [tc–Mg]+insertion into the binding tunnel, ring A of the tc molecule is anchored by hydrogen bonds to different residues including His64, Asn82, Phe86 and Gln116 (Fig 3) His64 is involved in the conforma-tional change of TetR associated with induction [14] and its interaction with tc fixes the C terminus of helix a4 This state
is stabilized by a network of cooperative hydrogen bonds including a chain of eight water molecules (Fig 5) that is not found in the free form of TetR Val57 participates in this so-called water zipper [30], representing the only
Table 2 In vivo data and thermodynamic stabilities of different TetR variants.
b-gal [%] b-gal [%] Urea 1/2 [ M ] Urea 1/2 [ M ] DUrea 1/2 [ M ] Urea 1/2 [ M ] DUrea 1/2 [ M ]
a
Induction was determined at 0.2 lgÆmL)1atc The 100% expression of b-galactosidase corresponds to 10920 ± 1451 units.bUrea 1/2 -values of chimeric TetR variants were calculated in the absence of the inducer atc by the change of the fluorescence at 330 nm or in the presence of atc from the change of the energy transfer signal at 515 nm for tc and 545 nm for atc excited at 280 nm detected at 28 C.
Table 3 Mg2+-independent (K T ) and -dependent (K A ) binding constants, association and calculated dissociation rate constants of atc to TetR variants.
K T
[· 10 7
M )1 ]
K A
[· 10 11
M )1 ]
k ass
[· 10 6
M )1 Æs)1]
k dissa
[· 10)6s)1]
a Calculated as k diss ¼ k ass /K A
Trang 8nonconserved residue of the contacting amino acids (in
Fig 1B marked by›) The assumption that the exchange to
Ile could lead to a distortion of the arrangement in this
water network connecting the helixes a4 and the loop
between helices a6 and a7 is in agreement with the strong
decreased complexstability of this single mutation alone
Although Ala61 is not directly involved in interacting
with the water zipper the exchange to Asp could sterically
influence residue 59 coordinating water W6 due to the larger
size and the introduction of a charged residue
From the thermodynamic point of view, this different
stabilization of TetR(B) and TetR(D) should be reflected in
their equilibrium association constants KA However,
cal-culatingDG from the determined binding constants KAby
DG ¼) RT*lnKA reveals 65.5 kJÆmol)1for TetR(B) and
68.9 kJÆmol)1for TetR(D) Taking into account the stability
of the free proteins [25] leads to complexstabilities of
140 kJÆmol)1 for TetR(B)[atc–Mg]2 and 123 kJÆmol)1
TetR(D)[atc–Mg]2, respectively Only the value for
TetR(D)[Mg-atc]2resembles the result from the
denatura-tion experiment This discrepancy for TetR(B) could be
explained by an enthalpy–entropy compensation effect
taking into account that amino acid replacements alter
both enthalpy and entropy contributions to ligand binding
As shown for the rat intestinal fatty acid-binding protein the
changes in molecular interactions may not necessarily
correlate with changes in affinity [31,32] The two identified
residues responsible for the different complexstability of TetR(B) and TetR(D) with atc belong to an internal water network which could be partially destroyed by the replace-ment to the respective residues of TetR(B) This fact might lead to the consequence that the local conformational flexibility of the ligand recognition site is increased due to the observation that binding of buried structural water molecules increase flexibility [33] This increased flexibility leads to an increase of entropy, which is probably not the case in TetR(B) due to a disordered water network organization This compensation explains the reduced DGU(H2O) value for TetR(B)[atc–Mg]2 deduced from urea-induced denaturation
The water zipper could be part of a functional epitope Taking into account that interactions between biological molecules cannot be reduced to the description of static molecular structures the function of a protein depends also
on the distribution and the populations of its conforma-tional states [34] Such a mechanism provides multiple pathways and allows a single molecular surface to interact with numerous structurally distinct binding part-ners, accommodate mutations through shifts in the dynamic energy landscape and as such is evolutionarily advantageous [9]
Although we are just beginning to understand the properties that makes these consensus binding sites unique, the role of conformational changes induced upon binding at the protein interface has emerged as a factor of key importance Because this separation/discrepancy is not seen for tc it is most likely that the water zipper plays an important role for stabilization and ligand affinity but not for induction This arrangement therefore mediates an indirect recognition mode of TetR This points out that internal bound water molecules increase protein flexibility which is responsible for specificity of ligand binding These findings contribute to the basic knowledge for drug design necessary to improve specificity of the TetR system
Acknowledgements
We thank Prof F X Schmid (University Bayreuth) for helpful and stimulating discussions and Dr Oliver Scholz for help in calculating the binding constants This work was supported by the Deutsche Forschungsgemeinschaft through SFB 473 and the Fonds der Chemischen Industrie.
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