This journal is c The Royal Society of Chemistry 2011 Mol. BioSyst.
Cite this: DOI: 10.1039/c1mb05281k
Minor groove recognition is important for the transcription factor
PhoB: a surface plasmon resonance study
M. Ritzefeld,aK. Wollschlager,wa G. Niemann,
aD. Anselmetti
band N. Sewald*
a
Received 5th July 2011, Accepted 19th August 2011
DOI: 10.1039/c1mb05281k
The two-component regulatory system PhoR/PhoB induces the expression of several genes in
response to phosphate starvation in Escherichia coli. In order to quantify these protein–DNA
interactions and to study the time-resolved dynamics of the binding mechanism, the specific
recognition of different oligonucleotide duplexes by the DNA-binding domain of PhoB
(PhoBDBD) was analyzed using surface plasmon resonance. In addition the two point mutants
PhoBDBDD196A and PhoBDBDR219A were obtained and the DNA recognition in comparison to
the wildtype PhoBDBD was investigated. Aspartic acid 196 and arginine 219 mediate specific
minor groove interactions. All results reveal that at high PhoBDBD-concentrations all recognition
sequences of the pho box are occupied. Decreasing the protein amount results in a mixture of free
oligonucleotides and DNA molecules occupied by two WT-PhoBDBD. Moreover, the SPR results
indicate that both binding site segments, the TGTCA-motif and the A/T-rich minor groove,
are essential for the binding process. A comparison of different regulons additionally proved the
dependency of the recognition process on the base composition of the minor groove.
Introduction
Most microorganisms are exposed to constantly changing
environmental conditions. Two-component regulatory sys-
tems (TCRSs) enable bacteria to sense these alterations and
to adapt cellular processes by regulating protein expression. In
Escherichia coli the PhoR/PhoB TCRS senses the environ-
mental phosphate concentration and regulates the response to
phosphate starvation.1 The transcriptional activator PhoB is
composed of two domains, the N-terminal regulatory domain
(residues 1–127) and the C-terminal DNA-binding domain
(PhoBDBD, residues 127–229).2
Inactive PhoB exists in a monomer–dimer equilibrium. The
DBDs of the inactive dimer point towards opposite directions.
An external phosphate concentration below 4 mM causes the
inner-membrane histidine kinase PhoR to activate PhoB by
phosphorylation.1,3 In the active state, the receiver domains
(PhoBRD) of two response regulators form a two-fold sym-
metric dimer with both DBDs orientated in a parallel head to
tail arrangement.3,4 This structural change enables PhoB to
bind to the pho box, a DNA sequence located 10 nucleotides
upstream of the �10 region in the regulon pho. The pho box
contains two TGTCA-motifs separated by an A/T-rich
region.1,2 Like other winged helix-turn-helix proteins, the
DBD of PhoB contains a recognition helix (a3, residues
192–206), a second helix that stabilizes the protein–DNA
complex (a2, residues 176–184) and a C-terminal b-hairpinthat interacts with the A/T-rich minor groove (cf. Fig. 1). A
loop, called the transactivation loop, which replaces the turn
motif, recruits thes70 subunit of the bacterial RNApolymerase.2,5
To date, 31 genes regulated by nine different pho regulons are
known to be directly controlled by the TCRS PhoR/PhoB.6
The corresponding sequences of the nine transcriptional units
are compiled in Table 1.
Although the structures of the DBD and the regulatory
domain have been determined by X-ray and NMR, only a few
Fig. 1 Crystal structure of two PhoBDBD-proteins bound in a head to
tail arrangement to an 18 bp double-stranded cognate recognition
sequence. : helix a1; : helix a2; : transactivation loop;
: helix a3 (DNA recognition helix); : b-sheets b6 and b7
(C-terminal b-Hairpin).2
aOrganic and Bioorganic Chemistry, Bielefeld University,PO Box 100131, 33501 Bielefeld, Germany.E-mail: [email protected]; Fax: +49 521 106 8094;Tel: +49 521 106 2051
b Single Molecule BioPhysics & Systems NanoBiology,Bielefeld University, Bielefeld, Germany
w New address: MorphoSys AG, Martinsried/Planegg, Germany.
MolecularBioSystems
Dynamic Article Links
www.rsc.org/molecularbiosystems PAPER
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quantitative results concerning the dynamics of the PhoB–DNA
interaction have been reported.7–11 Recently we analyzed
peptide and protein epitopes of the DBD of PhoB with respect
to DNA binding at the single molecule level using atomic force
microscopy (AFM) and dynamic force spectroscopy. We were
able to determine kinetic data, such as the thermal dissociation
rate constants. Furthermore we performed an alanine scan to
reveal the contributions of certain amino acid residues to the
binding process.7,8 To date only three investigations of
the equilibrium dissociation constant KD of PhoB–DNA
complexes have been reported.9–11 Makino et al. used DNase I
foot-printing in order to analyze the interactions between the
DBD of PhoB and the pst-promoter. The authors identified
the DNA binding domain of PhoB and the two pho boxes in
the regulon. Moreover the findings of Makino et al. revealed
that the complex consisting of PhoBDBD and pst exhibits an
equilibrium dissociation constant of 0.51 mM.9 McCleary
analysed the interactions between phosphorylated and unpho-
sphorylated PhoB and a synthetic 30 bp DNA containing one
pho box using a gel mobility shift assay. Due to phosphoryla-
tion the affinity of PhoB was increased by a factor of 10.10
Ellison and McCleary used fluorescence anisotropy measure-
ments in order to determine the dissociation constant of a
PhoBDBD–DNA complex. The corresponding data indicate
that the complex consisting of unphosphorylated full-length
PhoB, PhoBDBD and a synthetic double-stranded hairpin with
one DNA binding motif exhibits an equilibrium dissociation
constant of 0.44 and 0.06 mM respectively.11
Surface Plasmon Resonance (SPR) is a powerful method to
study time-resolved dynamics of biomolecular interactions.
One binding partner is immobilized on a chip surface. Binding
of the other molecule is then detected as a change of the
refractive index which corresponds to a change in mass. One
big advantage of SPR is the fact that the interaction can be
monitored very accurately in real time. Unspecific interactions
can be discriminated during the measurement from the specific
binding without the need to perform additional competition
experiments. Furthermore, a low abundance of both bio-
molecules is necessary and three interaction partners can be
analyzed simultaneously.12 Here we report the results
we obtained from the analysis of the interaction between
PhoBDBD and several DNA duplexes using SPR. Oligonucleo-
tides of different lengths containing one or two recognition
sequences based on the pst-regulon were used to determine the
binding mechanism of PhoBDBD.
Phosphate starvation induces among others the expression
of the genes phoA, phoH, pstS, pstC, pstA, pstB and phoU.
phoA encodes the periplasmic alkaline phosphatase.13 The
function of the protein encoded by phoH is still undefined,
though it has been shown to be an ATP binding protein and is
considered as RNA helicase.14,15 The genes pstS, pstC, pstA,
pstB and phoU encode the periplasmic phosphate-binding
protein (PstS), integral membrane proteins that mediate
translocation of phosphate across the inner membrane (PstC,
PstA), an ATP binding protein that supports the transport
process (PstB), and a protein that does not participate in the
translocation (PhoU), respectively.13,16–18 All of these genes
are part of the pst-operon and regulated by two pho boxes
upstream of the pstS gene. The operon is transcribed in its
entirety in response to phosphate starvation.19 A comparison
of the promoters of phoA, phoH and the pst-operon indicates
that phoA and phoH contain one and the pst-operon two pho
boxes (cf. Table 1).17,18 The sequences of these recognition
sites mainly differ in the A/T-rich minor grooves. All direct
contacts with bases are mediated by aspartic acid 196 and
arginine 219 (cf. Fig. 2).
In order to quantify the influence of the minor groove and
its base composition on the PhoBDBD recognition process,
binding of the PhoBDBD towards different dsDNA molecules
based on the pst, phoA and phoH pho box sequence was
analyzed.
Results and discussion
We investigated the contributions of the A/T-rich minor
groove sections by analyzing the binding of PhoBDBD towards
different 18, 24 and 40 bp dsDNA molecules (cf. Table 2).
Moreover, the interactions between the wildtype protein
and different 24 bp DNA molecules based on the pho box
Table 1 Sequences of the nine different transcriptional units recog-nized by the PhoR/PhoB TCRS.2,6 The three promoter sites comparedhere are printed in bold
phnCDEFGHIJKLMNOP C TGTTA GTCAC T TTTAA TTAAC
phoA C TGTCA TAAAG T TGTCA CGGCC
phoBR T TTTCA TAAAT C TGTCA TAAATphoE C TGTAA TATAT C TTTAA CAATCphoH C TGTCA TCACT C TGTCA TCTTT
eda C TTGCG TGAAA A ACTGT CCGGTpstSCAB-phoU C TGTCA TAAAA C TGTCA TATTC
C TTACA TATAA C TGTCA CCTGT
ugpBAECQ C CGTCA CCGCCT TGTCA TCTTT C TGACA CCTTAC TATCT TACAA A TGTAA CAAAAA AGTTA TTTTC C TGTAA TTCGA
psiE G TTGAA CAAAA C ATACA CAAAAA ATATA GATCT C CGTCA CATTT
Consensus C TGTCA TAAAX C TGTCA CAXTXFig. 2 Overview of the interactions between PhoBDBD and the minor
groove regions between the TGTCA-motifs in the pho box. lines
indicate the interactions found in the crystal structure by Blanco et al.
Contacts found only in the refW structure are given in .
lines indicate contacts detected in both structures.2,5
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sequences of the regulons of the pst-operon and the genes
phoH and phoA were compared using SPR (cf. Table 2). To
elucidate the binding contributions of the amino acids directly
interacting with bases of the minor groove sequence, aspartic
acid 196 and arginine 219 were mutated to alanine and the
changes in affinity were determined. PhoBDBDD196A and
PhoBDBDR219A were obtained by site-directed mutagenesis
from PhoBDBD and overexpressed in E. coli as fusion proteins
with an intein sequence and a chitin binding domain. The
wildtype (WT) and the mutated proteins were purified by
intein-mediated protein purification.7 Structural analysis of
all proteins and the DNA molecules was carried out using
circular dichroism spectroscopy (CD). Moreover, structural
changes upon binding of WT-PhoBDBD and PhoBDBDR219A
to dsDNA were investigated using CD spectra of the corres-
ponding DNA–protein complex.
SPR-analysis
Binding was investigated in real time using SPR to gain more
details of the recognition process. The different 50-biotinylated
dsDNA molecules were immobilized on a streptavidin sensor
chip surface. Random dsDNA of the same length was
immobilized to the reference cell to eliminate response differences
caused by nonspecific binding (cf. Table 1).
Short oligonucleotides consisting of 12 bp were initially
analyzed to determine the appropriate ligand size. No specific
recognition was observed in the case of the 12 bp dsDNA
(data not shown). Normally, three to six additional bases on
both sides of the recognition sequence are required as
spacers.12 Therefore, longer oligonucleotides composed of
18 to 40 bp were used.
Prior to immobilization all annealing solutions were
analyzed by gel filtration. About 400 RU (0.4 ng�mm�2) of
the respective DNA molecule was immobilized on the
SA-Chip. Normally, a smaller loading would be required to
obtain an analyte response of less than 100 RU in order to
prevent mass transport limitations.12 In the course of the
systematic optimization of the experiment smaller surface
concentrations were tested, but gave unsatisfactory results
(data not shown).
Analysis of DNA molecules based on the sequence of the
pst-regulon
The Biacore evaluation software in principle allows the
extraction of kinetic parameters and the dissociation constant
by fitting response curves to different kinetic models using a
nonlinear least-square algorithm.20 Due to the fast association
and dissociation of PhoB all SPR curves are characterized by a
steep slope at the beginning and at the end of the injection
(cf. Fig. 3). Direct analysis using the BiaEvaluation software
did not result in a reliable fit. Therefore, all dissociation
constants KD were determined using the equilibrium response
at different ligand concentrations. Calculation of the KD
values was then achieved using nonlinear regression to fit the
saturation curves to a two- or a one-site binding model
(cf. Fig. 3h and i). Each measurement was performed twice.
Sensograms (d)–(g) in Fig. 3 display both runs of each analyte
concentration (coloured in black and red) to demonstrate the
reproducibility of the method.
In the case of the complex consisting of WT-PhoBDBD and
MmX 18 (B) a one-site binding model was used to estimate the
dissociation constant as 14.5 mM. A comparison of the SPR
curves of MmM 18 (A) and MmX 18 (B) reveals that the
response signals of the DNA with two TGTCA-motifs (A) are
twice as high as the signals regarding to the DNA containing
one TGTCA-motif (B) (cf. Fig. 3a and b). These findings
prove the specific recognition of the major groove sequence
(TGTCA-motif), for two WT-PhoBDBD-proteins are able to
bind to MmM 18 (A) simultaneously. The one-site binding
model could only be used to fit the data; all other models
failed to do so. Therefore, the one-site binding model is the
only suitable model to characterize the formation of the
WT-PhoBDBD-MmM 18 (A) complex and gave the corres-
ponding KD of 17.8 mM. In consideration of the confidence
intervals of the two complexes, these results indicate that
binding of WT-PhoBDBD to MmX 18 (B) and MmM 18 (A)
exhibits comparable dissociation constants, respectively. This
apparently contradictory aspect will be discussed below.
A 40 bp oligonucleotide containing one binding site (K)
was analyzed to exclude that higher flexibility of the oligo-
nucleotides might influence the equilibrium dissociation constant.
Table 2 Sequences of all 50-biotin-labeled or unlabeled oligonucleotides. All DNAmolecules correspond to the sequence of the pst-regulon exceptfor phoA (I) and phoH (J). The TGTCA-motif is printed in bold and the A/T-rich minor groove in italics. Nomenclature of the oligonucleotides:M = Major groove (TGTCA-motif), m = minor groove (A/T-rich sequence), X = major groove random sequence, x = minor groove randomsequence
bp Nr. Name Sequence (50 - 30)
18 A MmM 18 CTGTCATAAAACTGTCATB MmX 18 CTGTCATAAAACCGGATCC XmM 18 CGAGGCTAAAACTGTCAT
24 D MmMx 24 CTGTCATAAAACTGTCAAGCATCTE MmMm 24 CTGTCATAAAACTGTCATATTCCTF MmXx 24 CTGTCATAAAACGAGGCAGCATCTG XmMx 24 CGAGGCTAAAACTGTCAAGCATCTH XmMm 24 CGAGGCTAAAACTGTCATATTCCTI phoA CTGTCATAAAGTTGTCACGGCCGAJ phoH CTGTCATCACTCTGTCATCTTTCC
40 K Mm 40 TCAGACTGAAGACTTTATCTCTCTGTCATAAAACCGGATC18 L random 18 CGAGGCAGCATACGGATC24 M random 24 CGAGGCAGCATACGGATCCGAGGC40 N random 40 CGAGGCAGCATACGGATCCGAGGCAGCATACGGATCCGAG
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The corresponding dissociation constant of 20.1 mM shows
that elongating pst-oligonucleotides in the 50 directory does
not affect the affinity (cf. Fig. 3c and h).
Although the 18 bp sequence 50-CTGTCATA(A/T)A(T/A)-
CTGTCA(C/T)-30 is always termed pho box in the literature,
the minor groove sequence of the second binding site in
position 30 is missing. Therefore, we analyzed 24 bp oligo-
nucleotides that contain two complete binding sites to
extend the results of the 18 bp duplexes and to validate the
relevance of the minor groove region for the recognition process.
Fig. 3 (a)–(g) Real-time analysis of different oligonucleotides based on the sequence of the pst-Regulon. The protein WT-PhoBDBD was used as
an analyte at different concentrations between 30 and 0.5 mM and the oligonucleotide duplexesMmM 18 (A),MmX 18 (B),Mm 40 (K),MmMx 24 (D),
MmMm 24 (E),MmXx 24 (F) and XmMm (H) as ligands. Every measurement was performed twice. Sensograms (d)–(g) display both runs of every
analyte concentration coloured in black and red, to show the reproducibility of the method. (h)–(i) Saturation curves were obtained by plotting
the response unit at equilibrium of different analyte-concentrations. (j) The dissociation constants were calculated using nonlinear regression
(CI = 95% confidence interval).
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The response signals corresponding to the DNA with two
TGTCA-motifs (E) are again twice as high as the signals
corresponding to the DNA containing only one TGTCA-
motif (F) (cf. Fig. 3e and f). In the case of MmXx 24 (F) the
dissociation constant was estimated as 21.9 mM using a
one-site binding model. These results indicate that the two
complexes consisting of WT-PhoBDBD and either MmX 18 (B)
or MmXx 24 (F) are both characterized by a comparable
dissociation constant, respectively. Thus elongating the DNA
with a random DNA sequence does not affect the binding
mechanism. In the case of the specific recognition of MmMm
24 (E) fitting the equilibrium response units at different
concentrations to a two-site binding model did not give reliable
results, although the DNA duplex contains two binding sites and
the response signals are twice as high as the signals regarding to
MmXx 24 (F). Therefore, the equilibrium dissociation constant
was estimated as 1.4 mM (cf. Fig. 3h and i) using a one-site
binding model.
Binding of WT-PhoBDBD towards MmMx 24 (D) was inves-
tigated (cf. Fig. 3d and h), to further analyze the difference
between the protein–DNA complexes containing MmM 18 (A)
and MmMm 24 (E) respectively. The corresponding oligo-
nucleotide-complex exhibits a dissociation constant of 25.1 mMthat was determined using a one-site binding model. Therefore the
protein–DNA complexes consisting of WT-PhoBDBD, MmM 18
(A) and MmMx 24 (D) show a similar recognition behavior. In
conclusion, elongating MmM 18 (A) using an arbitrary minor
groove sequence does not affect the recognition process.
In order to investigate the role of the minor groove and
whether there is a difference in affinity between the two binding
sites in positions 50 and 30, a 24 bp DNA containing a major
and a minor groove in position 30 and another A/T-rich minor
groove in position 50 (H) was analyzed. The complex consisting
of WT-PhoBDBD and XmMm 24 (H) exhibits a dissociation
constant of 14.5 mM. The fact that only one WT-PhoBDBD is
able to bind toXmMm 24 (H) reveals that the 50 A/T-rich minor
groove alone is not recognized by WT-PhoBDBD. Comparing
these results with the data obtained for MmM 18 (A) emphasizes
the relevance of the TGTCA-motif for the specific binding process,
for two WT-PhoBDBD recognize the 18 bp dsDNA.
Moreover, the dissociation constants of the complexes
consisting of WT-PhoBDBD, MmX 18 (B), MmXx 24 (F),
Mm 40 (K) and XmMm 24 (H), respectively, are comparable.
Hence, both binding sites (each with a major and minor
groove) in positions 50 and 30 of the pho-box exhibit equal
affinities for WT-PhoBDBD.
The analysis of oligonucleotides containing only binding site
fragments based on the sequence of the pst-regulon was
expected to provide further information on the contributions
of the A/T-rich minor groove and the major groove. There-
fore, the interactions between WT-PhoBDBD and XmM 18 (C)
and XmMx 24 (G), respectively, were investigated. In both
cases no specific binding was observed (data not shown). All
these results corroborate the relevance of the specific 30 minor
groove recognition. The TGTCA-motif lacking the appropriate
30 minor groove sequence is insufficient for specific recognition
on its own. Even elongating the sequence using an arbitrary
minor groove (XmMx 24 (G)) solely results in unspecific
binding. Furthermore, these results indicate again that minor
groove binding without addressing the major groove is also
not sufficient. Only the presence of both sections in the right
arrangement enables specific interactions. In the case of
MmM 18 (A) binding to the recognition site that exclusively
consists of the major groove is possible and leads to complexes
consisting of two proteins and the dsDNA. One possible
explanation for the disparity between XmMx 24 (G) and
MmM 18 (A) concerns protein–protein interactions. The
second WT-PhoBDBD bound to the neighboring recognition
site compensates the missing minor groove interaction in the
case of MmM 18 (A).
Interaction of PhoBDBD
point mutants with MmMm24 (E)
Aspartic acid 196 and arginine 219 that mediate the involved
specific interactions were replaced by alanine (PhoBDBDD196A
and PhoBDBDR219A) to further elucidate the recognition
process between PhoBDBD and the A/T-rich minor groove.
SPR measurements were performed using both mutants as
analytes and oligonucleotide duplex MmMm 24 (E) as a
ligand. Although each protein still specifically recognizes the
pho box in the major groove (very weak SPR responses can be
detected), DNA binding is almost abolished in both cases
(cf. Fig. 4). These results are supported by the circular
dichroism data (cf. Fig. 6h). In conclusion, the specific inter-
actions between aspartic acid 196, arginine 219 and the minor
groove sequence are essential for the binding process.Manipulating
the minor groove recognition of PhoBDBD by changing the
base composition of the A/T-rich sequence or by replacing the
protein residues, involved in minor groove contacts, significantly
affects the entire DNA binding.
Interaction of WT-PhoBDBD with DNA molecules based on the
sequence of different pho Regulons
A qualitative comparison of the SPR sensograms reveals that
the response signals corresponding toMmMm 24 (E) are twice
as high as the signals corresponding to phoA (I) and phoH (J),
respectively (cf. Fig. 5). Furthermore, the maximum response
unit differences of the latter resemble the results of the dsDNA
containing one single recognition site (Fig. 3, only one major
and one minor groove; oligonucleotides B, F, H, K). The
dissociation constants confirm the observed tendencies. The
complex consisting of phoA (I) and WT-PhoBDBD is charac-
terized by a dissociation constant of 7.1 mM although it
contains two binding sites with a major and a minor groove.
This value is significantly higher than the dissociation constant
of the WT-PhoBDBD-pst (E) (KD = 1.4 mM) complex and
significantly lower than the values of the complexes containing
MmX 18 (B), MmXx 24 (F), XmMm 24 (H) and Mm 40 (K)
(KD = 14.5–21.9 mM) respectively. In the case of phoH (J),
which also contains two binding sites with the corresponding
major and minor groove sequences, the dissociation constant
was determined as 42.4 mM. This value has to be perceived
somewhat critically because of the large 95% confidence
interval from 15.0 to 80.6 mM. However, these data clearly
indicate that WT-PhoBDBD binds stronger to phoA (I) and the
pst-oligonucleotide (E) than to phoH (J). The affinity increases
from phoH (J) over phoA (I) to MmMm 24 (E). Analyzing the
minor groove in between the two TGTCA-motifs of all three
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regulons reveals that the pst-oligonucleotide exhibits an
A-tract sequence leading to a highly compressed groove.2
Comparing the base composition of this region among the
three regulons indicates that the A/T-content decreases from
100% for the pst-oligonucleotide (E) (50-AAAA-30) to 75% for
phoA (I) (50-AAAG-3 0) and 50% for phoH (J) (50-CACT-30;
cf. Table 2, Fig. 5e). In conclusion, the A/T-content of
the 30 minor groove sequences proportionally influences the
differences between the equilibrium dissociation constants
(see below).
CD spectroscopy
CD spectra of several oligonucleotide–protein complexes, the
DBD of PhoB and the corresponding pure oligonucleotides
were measured. All DNA molecules exhibit a positive band at
275 nm and a negative band at 248 nm with nearly comparable
intensities (cf. Fig. 6). The minimum is characteristic for right
handed B-DNA duplexes. The maximum is due to base
stacking.21,22 In conclusion, all oligonucleotides possess
equivalent conformational properties. In order to reveal
structural changes of the dsDNA upon binding of both
PhoBDBD molecules (WT-PhoBDBD and PhoBDBDR219A)
differential CD spectra were calculated by subtracting the
corresponding protein spectra from the protein–DNA spectra.
We recently reported structural changes upon binding of
WT-PhoBDBD to MmM 18 (A) and concluded that the duplex
is bent upon complex formation (cf. Fig. 6a).7 These data are
supported by NMR and X-ray structure analysis.2,5 For the
DNA molecules with only one binding site (MmXx 24 (F),
XmMm 24 (H)) the CD spectra did not change upon addition
of pure dsDNA. The same results were observed for the 24 bp
random DNA (M) respectively (cf. Fig. 6c, d and g). Taking
into consideration that WT-PhoBDBD is able to bind to one
Fig. 5 (a)–(c) Real-time analysis of oligonucleotides based on the sequence of different pho-Regulons. The protein WT-PhoBDBD was used as an
analyte at different concentrations between 30 and 0.5 mM and the oligonucleotide duplexes MmMm 24 (E), phoA (I) and phoH (J) as ligands. (d)
Saturation curves were obtained by plotting the response unit at equilibrium of different analyte-concentrations. (e) The dissociation constants
were calculated using nonlinear regression (CI = 95% confidence interval).
Fig. 4 Analysis of the interaction between PhoBDBD point mutants andMmMm 24 (E). The oligonucleotide duplexMmMm 24 (E) was used as a
ligand. The proteins (a) WT-PhoBDBD, (b) PhoBDBDD196A and (c) PhoBDBD R219A were used as analytes respectively.
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single binding site in SPR real-time experiments, these
data imply that the affinity is either too small, or that
binding of two proteins is necessary to induce conformational
changes.
For the 24 bp DNA MmMm 24 (E) addition of one, two or
three equivalents of WT-PhoBDBD results in a significant
change of the CD (cf. Fig. 6b). Both bands are increased
compared to pure dsDNA. Moreover the positive band is
red-shifted from 275 to 277 nm. Comparing these differences
at distinct ratios reveals that the maximum and the minimum
increase from zero to one to two PhoBDBD-equivalents.
However, more than two equivalents of protein do not lead
to further changes of the CD spectrum (cf. Fig. 6b). These
alterations at different protein ratios were observed for MmM
18 (A), too. Taking into consideration that bending of the
DNA is not detected in the case of oligonucleotides possessing
one single binding site (MmXx 24 (F) and XmMm 24 (H)),
these data indicate that even at a protein : DNA ratio of 1 : 1
two WT-PhoBDBD-molecules have to bind to one DNA
molecule (see below).
Although the oligonucleotidesMmMm 24 (E) (pst), phoA (I)
and phoH (J) exhibit two complete binding sites with two
major and two minor grooves, the circular dichroism spectra
of phoA (I) and phoH (J) display less pronounced concentration-
dependent changes compared to the pst-oligonucleotide
MmMm 24(E) (cf. Fig. 6b, e and f). In the case of phoA (I)
only the positive band is increased upon complex formation.
The structural changes indicated by the CD results upon
binding of WT-PhoBDBD to phoH (J) are negligible (cf. Fig. 6f).
These results demonstrate in good agreement with the SPR
data, the dependence of the degree of the oligonucleotide
modification and the associated protein–DNA affinity on the
sequence of the corresponding recognition site.
In order to determine the contributions of certain amino
acids involved in the binding of the minor groove sequence,
aspartic acid 196 (PhoBDBDD196A) and arginine 219
(PhoBDBDR219A) were mutated to alanine. CD spectra of
all three proteins were compared to exclude structural
differences between WT-PhoBDBD and both mutant proteins
(cf. Fig. 6i). The results obtained are in good agreement with
Fig. 6 (a)–(h) Differential CD-spectra of PhoBDBD-DNA complexes minus protein spectra at different ratios (colour code: unbound Pho box
DNA/ / / ). (i) CD spectra of wild-type PhoBDBD and the mutants PhoBDBDD196A and
PhoBDBDR219A.
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the CD spectra published recently and reveal that all proteins
exhibit similar structures.7 Differential CD spectra of the
PhoBDBDR219A-MmMm 24 (E) complex at different ratios
were obtained (cf. Fig. 6h), to elucidate the effect of the point
mutation on the binding process. No differences in compar-
ison to the results of the pure DNA duplex were detected.
These results are in good agreement with the SPR-data that
also show a nearly complete loss of affinity of the two point
mutants for MmMm 24 (E) (cf. Fig. 4).
The WT-PhoBDBD
-binding mechanism
Comparing the resonance units in equilibrium between oligo-
nucleotides that contain only one binding site (one major and
one minor groove; e.g. MmXx 24 (F)) with dsDNA of the same
length that contains two binding sites (two major grooves and
two corresponding minor grooves; e.g. MmMm 24 (E)) reveals
that the signals of the latter are twice as high as the resonance
units of the first ones (cf. Fig. 3e and f). Consequently, two
PhoBDBD molecules bind to a complete pho box (e.g. MmMm
24 (E)) and only one WT-PhoBDBD recognizes a single binding
site (e.g. MmXx 24 (F)). The corresponding dissociation
constant of the 2 : 1 protein:dsDNA interaction (KD =
1.4 mM) is thereby reduced by a factor of 10–20 in comparison
to the 1 : 1 protein : dsDNA interaction (KD = 21.9 mM).
Therefore the two protein moieties seem to stabilize the
ternary protein–DNA complex amongst others by protein–
protein interaction, which is in good agreement with the
crystal structure analysis results.2 However, the SPR-results
indicate that binding of WT-PhoBDBD to e.g. MmMm 24 (E)
should be described using a one-site binding model with
neither positive, nor negative cooperativity. According to the
circular dichroism experiments the dsDNA is already
bent at a protein : DNA ratio of 1 : 1 in the case of the
WT-PhoBDBD-MmMm 24 complex (cf. Fig. 6b), while no
conformational change is observed for dsDNA with only
one specific binding site (cf. Fig. 6c and d). Therefore, we
suggest that both recognition motifs of the pho box are
occupied by PhoBDBD at high PhoBDBD concentration and
that this binding event is non-cooperative. Decreasing the
protein amount to a protein : DNA ratio of 1 : 1 and below
results in an equilibrium mixture of free dsDNA and dsDNA
occupied by two WT-PhoBDBD. In the case of a stepwise
association mechanism operative at low WT-PhoBDBD con-
centration, each dsDNA e.g. MmM 18 (A) andMmMm 24 (E)
were expected to be solely occupied by a single protein. In this
case, no bending would be observed in the CD spectra at a
protein : DNA ratio of 1 : 1. An increase of the WT-PhoBDBD
concentration would bring about an association event leading
to 2 : 1 protein : dsDNA complexes. This additional binding
event (binding of a second PhoBDBD to the existing 1 : 1
complex) demands SPR-data evaluation using a two site
binding model, which was not applicable in all cases examined.
An alternative explanation involves a monomer–dimer
equilibrium of PhoBDBD already in solution. The regulatory
domains of full-length PhoB normally mediate dimerization.
Although these regions are missing in the case of WT-PhoBDBD,
extensive protein–protein contacts of two PhoBDBD molecules
were proven by crystal structure analysis.2–4 One argument
against this hypothesis is that gel filtration experiments did
not indicate the existence of a PhoBDBD dimer in solution
(data not shown).
Another main conclusion from the SPR- and the CD-results
concerning the oligonucleotides of the pst-regulon regards the
relevance of the minor groove recognition. In particular
all data obtained for the oligonucleotides XmM 18 (C),
MmMm 24 (E) and XmMx 24 (G) reveal that minor groove
contacts are essential for the overall recognition. These
conclusions are supported by the results concerning the two
mutants PhoBDBDD196A and PhoBDBDR219A.
Variations of the nucleotide sequence by using different
pho-regulons further indicate a connection between the
A/T-content of the central minor groove and the dissociation
constant. Two different explanations take this tendency into
account:
Blanco et al. proposed that DNA bending is probably
determined by the nucleotide composition of the A/T-rich
region.2 Bending of the oligonucleotide might enhance the
DNA–protein interactions by creating a more favorable
DNA-surface. Comparing the changes in the CD-effect at
different protein : dsDNA ratios between the three regulons
reveals that the oligonucleotide possessing the sequence of
the pst-regulon (E) is bent stronger in comparison to the
DNA-duplexes exhibiting the sequence of phoA- (I) and even
more than the phoH-regulon (J).
The second explanation involves the binding contribution of
the amino acids directly interacting with the bases of the minor
groove. Each base pair in the major groove of an oligonucleo-
tide has a unique hydrogen-bonding signature facilitating the
specific recognition of the corresponding nucleotide sequence
by proteins. The minor groove often is considered to lack this
signature.23 However, the minor groove may act as a hydrogen
bond donor and acceptor. Hence, it is well-known to be a
target for specific interactions, e.g. with distamycin-type
heterocycles.24 According to Rohs et al., the width of the
minor groove and, consequently, the negative electrostatic
potential govern recognition of the minor groove and prevail
over hydrogen bonding. Positively charged amino acids,
mainly arginine, interact with these narrow grooves. In con-
clusion specific recognition of the minor groove is warranted
by the variation of the DNA shape.23 Minor groove-narrowing
correlates with the base composition. A high A/T-content
favors narrow minor grooves whereas GC-rich regions are
more flexible and wider.25 In conclusion, the negative electro-
static potential of the central minor groove increases from
the pst-oligonucleotide (E) to the phoH-duplex (J) thereby
enhancing the corresponding interactions.
Two aspects contradict this explanation. First of all,
aspartic acid 196 plays a central role in the minor groove
recognition, too, but is negatively charged. Moreover, the base
composition of the 30-terminal minor groove of all three
regulons deviates from the A/T-content of the central minor
groove. Still, the difference concerning the dissociation
constants of the oligonucleotides MmMx 24 (D) and MmMm
24 (E) shows that the minor groove in position 30 is also
important for the recognition process. Another example
emphasizing this discrepancy concerns the oligonucleotides
MmMx 24 (D) and phoA (I). Comparing both minor groove
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sequences of the two oligonucleotides indicates that the central
minor groove exhibits an A/T-content of 100% in the case of
MmMx 24 (D) (50-AAAA-30) and 75% in the case of phoA (I)
(50-AAAG-30). A simultaneous trend is true for the terminal
minor groove:MmMx 24 (D) (50-GCAT-30) 50% and phoA (I)
(50-GGCC-30) 0%. However, the complex consisting of
WT-PhoBDBD and phoA (I) exhibits a lower dissociation
constant (KD = 7.1 mM) than the complex containing MmMx
24 (D) (KD = 25.1 mM) despite having a lower A/T-content.
Consequently, the A/T-content alone is insufficient to predict the
minor groove shape and the electrostatic potential. A similar
situation has been analyzed by Stefl et al.: solution structures of
the oligonucleotides d(GCAAAATTTTGC) and d(CGTTTTA-
AAACG) revealed that although the A/T-content of both
molecules is identical, the central ApT-step (highlighted in bold
figures) in the first oligonucleotide results in a local bend
towards the minor groove, whereas the central TpA-step
(highlighted in bold figures) in the second duplex locally bends
towards the major groove.25 Hence, TpA-steps prevent minor
groove narrowing by increasing their flexibility.23,25,26 One
approach to predict the sequence-dependent intrinsic flexibility
of the DNA is based on the empirical TRX (Twist, Roll and
X-disp) score,26 derived from NMR data in a solution of
several dinucleotide sequences. The higher the TRX score,
the greater the intrinsic flexibility is. For the minor grooves
described here, the TRX score shows a correlation between the
A/T-content of the minor grooves and the expected shapes
(cf. Table 3).26 With reference to the minor groove flexibilities
and the corresponding electrostatic potentials, the dissociation
constant of the complex containingMmMx 24 (D) would have
been predicted to be lower than the dissociation constant
regarding phoA (I).
In conclusion, minor groove narrowing and the corresponding
electrostatic potential is an important feature, influencing the
overall recognition process of the pho box by WT-PhoBDBD.
Still, other mechanisms including the interaction mediated by
aspartic acid 196 or the bending properties of the oligonucleo-
tide also have to be considered.
Experimental
E. coli strains were purchased from New England Biolabs
(Frankfurt a.M., Germany). All primers were purchased from
Eurofins MWG Operon (Ebersberg, Germany). Chemicals
were obtained from Sigma-Aldrich (Hamburg, Germany),
Acros (Geel, Belgium), or Applichem (Darmstadt, Germany).
MALDI-ToF mass spectra were recorded on a Voyager DE
instrument (PerSeptiveBiosystems, Weiterstadt, Germany)
with sinapinic acid as a matrix.
Protein expression and purification
Details concerning the preparation of WT-PhoBDBD and protein
purification have been reported elsewhere.7 The point mutants
D196A and R219A were both introduced into WT-PhoBDBD
by QuickChange site directed mutagenesis kit (Stratagene,
Amsterdam, Netherlands). Success of the mutation was
proven by DNA sequencing and MALDI-ToF MS of the
obtained proteins.
D196A. Forward primer:
GAAGACCGCACGGTCGCTGTCCACATTCGTCGC
Reverse primer:
GCGACGAATGTGGACAGCGACCGTGCGGTCTTC
R219A. Forward primer:
CATGGTGCAGACCGTGGCCGGTACAGGATATCG
Reverse primer:
CGATATCCTGTACCGGCCACGGTCTGCACCATG
Gel filtration
Gel filtration experiments were performed using an AKTA
Ettan (GE Healthcare, Munich, Germany) equipped with a
Superdex 75 3.2/30 PC column (GE Healthcare) at a flow of
40 mL min�1. Phosphate buffer (100 mM Na2HPO4, 150 mM
NaCl, pH 7.4) was used as an eluent.
DNA preparation
All oligonucleotides were obtained from Eurofins MWG
Operon (Ebersberg, Germany). The corresponding oligo-
nucleotide sequences are shown in Table 2. For SPR experi-
ments the 50 strain was biotinylated and the single stranded
DNA HPLC purified. The labeled or unlabeled forward
strands were annealed to their complementary reverse
strands by heating to 95 1C and gradually cooling to room
temperature. The annealing steps were performed in 20 mM
Tris-HCl, pH 7.4 containing 5 mM MgCl2 with an oligo-
nucleotide concentration of 10 mM. Success of hybridization
was analyzed by gel filtration subsequently.
CD spectroscopy
All CD spectra were recorded on a J-810 CD spectrometer
(Jasco, Groß-Umstadt, Germany) in a 1 mm quartz cell at
room temperature, using a scanning rate of 50 nm min�1, a
data pitch of 0.2 nm and three accumulations. Protein spectra
were recorded at a concentration of 12 mM. Initial DNA
concentration of 10 mM and initial protein concentrations of
0.2 mg mL�1 in phosphate buffer (10 mM Na2HPO4, 5 mM
NaCl, pH 7.4) were used to analyze structural changes in the
DNA–protein complexes. Protein solution or buffer was
added to the respective DNA solution to obtain ratios between
DNA and protein from 1 to 3. Molar CD absorptions were
calculated using eqn (1), where y is the ellipticity in milli-
degrees, c the final concentration in mol L�1 and l the cell path
length in cm.
De = Y/32980�c�l (1)
Table 3 A/T-content and TRX score of the internal minor groove(50) and the terminal minor groove (30) of MmMx 24 (D) andphoA(I)26
50-Minor groove 30-Minor groove
MmMx 18 (D)
50-AAAA-30 50-GCAT-30
A/T (%) 100 50TRX score 15 67
phoA (I)
50-AAAG-30 50-GGCC-30
A/T (%) 75 0TRX score 19 109
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In order to analyze structural changes upon protein–DNA
complex formation, spectra of the protein, the corresponding
DNA and the DNA–protein complex spectrum were recorded
at each concentration. In order to analyze the consequences of
protein–DNA interactions on DNA structure, the protein
spectrum was subtracted from the corresponding DNA–protein
spectrum.
SPR apparatus, surface preparation, DNA immobilization
All binding experiments were carried out on a Biacore 3000
(GE Healthcare, Munich). Streptavidin-coated sensor chips
were purchased from GE Healthcare (Munich, Germany). All
solutions were sterile-filtered before use. The protein dialysis
buffer was used as running buffer (100 mM Na2HPO4, 50 mM
NaCl, pH 7.4).
Prior to DNA immobilization the streptavidin surface was
primed three times. Then a solution consisting of 50 mM
NaOH and 1 M NaCl was injected for three times at a flow
rate of 20 mL�min�1 in order to prepare the surface followed by
two injections of regeneration solution (0.05% SDS in running
buffer). The surface was washed with buffer until a stable
baseline was obtained. In order to prevent inaccuracies due to
divergent reflection properties of the dextran surface, 70%
glycine was used to normalize the instrument. The system was
subsequently primed three times.
All biotinylated DNA duplexes were immobilized on the
streptavidin surface by injecting a 10 nM DNA solution at a
flow rate of 5 mL�min�1 until a RU difference of 400 was
detected. A control DNA with a random sequence equal in
length to the specific DNA (cf. Table 1, DNA L-N) was
immobilized on the first flow cell and used as a reference to
correct non-specific binding. Prior to the first measurement,
the chip was regenerated by injecting 0.05% SDS in running
buffer for two times and washed with buffer for five minutes.
SPR measurements
The chip surface was equilibrated with running buffer for three
minutes at 30 mL�min�1 prior to the injection of the sample.
Then 60 mL of the protein solution was injected during two
minutes. The dissociation phase was monitored for five
minutes, followed by regeneration with two injections of
15 mL of 0.05% SDS solution. Afterwards the surface was
washed with running buffer for five minutes. In all cases the
average of two measurements was used for data analysis. The
response units at equilibrium at different concentrations were
fitted using a one-site or a two-site binding model through
nonlinear regression (GraphPad Software, San Diego, USA).
Conclusions
In this study surface plasmon resonance and circular dichroism
spectroscopy were employed to obtain information on the
binding mechanism of PhoBDBD to its cognate recognition
sequence in the pst-, phoA and phoH-regulons. Both segments,
the TGTCA major groove motif and an A/T-rich minor
groove, are essential for the specific recognition of the pho
box. In the case of the pst-regulon two adjacent binding sites
enable binding of two PhoBDBD molecules to the regulon with a
dissociation constant reduced by a factor of approximately 10–20.
Moreover, CD results indicate that solely two complete
recognition sites containing both major and minor groove
residues enable PhoBDBD to significantly change the
conformation of the dsDNA. Dimerization of full-length
PhoB is mediated by the regulatory domains.3,4 These regions
are missing in the case of the WT-PhoBDBD used here, but
extensive protein–protein contacts of two PhoBDBD moieties
due to a good complementarity of the interacting surfaces were
already proven by crystal structure analysis. Regions involved
in PhoBDBD dimerization are the C-terminal tail and the
N-terminal b-sheet (b2–b4).2 Our results support these
findings and indicate that a mixture of free oligonucleotides
and DNA molecules occupied by two WT-PhoBDBD is formed
at low protein concentrations due to these protein–protein
interactions. The comparison of the pho box sequences of the
pst-operon and the regulons of phoA and phoH reveals that the
affinity of the transcriptional activator increases from phoH over
phoA to pst. The differential CD spectra confirm that the
composition of the minor groove is fundamental for the
recognition process. SPR measurements using PhoBDBDD196A
and PhoBDBDR219A as analytes support these results.
Recognition of the pho box is almost abolished upon replace-
ment of either of the two residues by alanine. Minor groove
narrowing and an increase of the corresponding negative
electrostatic potential play an important role. However, the
role of aspartic acid 196 or the bending properties of the
oligonucleotide sequence seem to influence the minor groove
recognition, too.
In conclusion, differences concerning the specific minor
groove recognition significantly affect the entire binding
process. Thus minor and major grooves are both of compar-
able significance for the recognition process.
Acknowledgements
This work was supported by the German Research Foundation
(DFG, SFB 613) and the German National Academic
Foundation (Studienstiftung des Deutschen Volkes, PhD
fellowship to Markus Ritzefeld).
Notes and references
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