Molecular Dynamic Article Links BioSystems...bind to the pho box, a DNA sequence located 10...

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

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www.rsc.org/molecularbiosystems PAPER

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Mol. BioSyst. This journal is c The Royal Society of Chemistry 2011

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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3 P. Bachhawat, G. V. T. Swapna, G. T. Montelione andA. M. Stock, Structure (London), 2005, 13, 1353–1363.

4 T. R. Mack, R. Gao and A. M. Stock, J. Mol. Biol., 2009, 389,349–364.

5 T. Yamane, H. Okamura, M. Ikeguchi, Y. Nishimura andA. Kidera, Proteins: Struct., Funct., Genet., 2008, 71, 1970–1983.

6 Y.-J. Hsieh and B. L. Wanner, Curr. Opin. Microbiol., 2010, 13,198–203.

7 K. Wollschlager, K. Gaus, A. Kornig, R. Eckel, S.-D. Wilking,M. McIntosh, Z. Majer, A. Becker, R. Ros, R. Anselmetti andN. Sewald, Small, 2009, 5, 484–495.

8 R. Eckel, S.-D. Wilking, A. Becker, N. Sewald, R. Ros andD. Anselmetti, Angew. Chem., 2005, 117, 3989–3993 (Angew.Chem., Int. Ed., 2005, 44, 3921–3924).

9 K. Makino, M. Amemura, T. Kawamoto, S. Kimura, H. Shinagawa,A. Nakata and M. Suzuki, J. Mol. Biol., 1996, 259, 15–26.

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17 F. Y. Chan and A. Torriani, J. Bacteriol., 1996, 178, 3974–3977.18 P. M. Steed and B. L. Wanner, J. Bacteriol., 1993, 175, 6797–6809.19 M. Aguena, E. Yagil and B. Spira, Mol. Genet. Genomics, 2002,

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Molecular Dynamic Article Links BioSystems...bind to the pho box, a DNA sequence located 10...

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