DnaK Dependence of the Mycobacterial Stress-Responsive Regulator
HspR Is Mediated through Its Hydrophobic C-Terminal Tail
Boudhayan Bandyopadhyay,aTwishasri Das Gupta,a* Debjani Roy,band Sujoy K. Das Guptaa
Department of Microbiology, Bose Institute, P1/12 C.I.T. Scheme VIIM, Kolkata, India,aand Department of Biophysics, Bose Institute, P1/12 C.I.T. Scheme VIIM, Kolkata,
eralotheractinobacteria,thisproteinissynthesizedfromthe dnaKJE-hspR operon.PreviousinvestigationsrevealedthatHspR
HspRs of Mycobacterium tuberculosis and related Actinomycetales therefore may have evolved to make these HspRs more sensi-
teins (HSPs), which include molecular chaperones that are re-
quired for the correct folding of the newly synthesized proteins as
well as denatured proteins (11). Although induction of HSP syn-
thesis is a universal process, the regulatory mechanisms that con-
trol their synthesis differ widely between organisms. One such
mechanism found in Escherichia coli involves the specific sigma
factor ?32, which is required for the recognition of heat shock
promoters by RNA polymerase (3). Under normal physiological
conditions, this sigma factor remains associated with DnaK and
therefore cannot bind to the promoter. Under heat shock condi-
tions, the denatured proteins which accumulate within the cell
titrate the DnaK away from ?32, leaving it free to function (32).
The sigma factor-dependent regulation of heat shock promot-
ers is not conserved in prokaryotes. Indeed, in many Eubacteria,
expression of heat shock genes and operons is controlled by spe-
cific repressors (14). In Bacillus subtilis, both the groEL and dnaK
binds to an inverted repeat named CIRCE (controlling inverted
repeat of chaperone expression) that is located in the regulatory
regions of these operons. The repressor activity of HrcA is posi-
tively modulated by GroEL. A titration model, similar to that in
the case of the DnaK/?32pair, has been proposed for GroEL/
is, however, a basic difference in the manners in which DnaK
regulates the activities of ?32and HrcA. In the case of ?32, it acts
of the repressor HrcA, it acts positively by assisting HrcA’s ability
to bind to CIRCE.
some Gram-negative bacteria, an additional repressor known as
he heat shock response is a tightly regulated process which is
induced under stress conditions (17). Induction of the heat
isms, there is a clear demarcation—HspR represses dnaK and
HrcA represses groEL. Interestingly, the activity of HspR, like that
of HrcA, is chaperone dependent. However, the chaperone in-
volved in this case is not GroEL but DnaK (2). HspR proteins of
Mycobacterium tuberculosis and several other organisms belong-
of the dnaKJE-hspR operon. The binding site of HspR, known as
HAIR (HspR-associated inverted repeats), is present upstream of
several genes and operons, including the one from which it is
synthesized. It has been proposed that expression from the
dnaKJE-hspR operon is controlled by a homeostatic feedback
mechanism involving HspR and DnaK (2). According to the pro-
posed mechanism, the operon is induced under heat shock con-
ditions, resulting in enhanced synthesis of DnaK as well as HspR.
When the amount of DnaK and HspR within the cell reaches a
critical level, they together bind to the operon promoter, thereby
In this study, the focus is on HspR from Mycobacterium tuber-
culosis. This organism is the causative agent of tuberculosis (TB).
A major problem with TB is chronic infection. This happens due
to the ability of M. tuberculosis to persist in an infected individual
for a prolonged period of time (33). The persistent form of TB is
key role in determining the fate of M. tuberculosis in the infected
Received 16 March 2012 Accepted 23 June 2012
Published ahead of print 29 June 2012
Address correspondence to Sujoy K. Das Gupta, email@example.com.
*Present address: Twishasri Das Gupta, Department of Cell Biology, Cleveland
Clinic Foundation, Lerner Research Institute, Cleveland, Ohio, USA.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jb.asm.org Journal of Bacteriology p. 4688–4697 September 2012 Volume 194 Number 17
individual. It was found that an HspR-deficient mutant of M.
and this boosted an immune response in the host against the
pathogen. Apart from the dnaKJE-hspR operon, the expression of
several other genes of M. tuberculosis is controlled by HspR. One
such gene which expresses the ?-crystallin family protein Acr2 is
required for pathogenesis (24). HspR therefore appears to play a
key role in controlling the expression of several M. tuberculosis
genes linked to pathogenesis.
been investigated in a previous study using HspR of Streptomyces
coelicolor, which, like M. tuberculosis, belongs to the phylum Acti-
nobacteria. In this study (2), it was found that DnaK can enhance
the binding activity of HspR in an ATP-independent manner.
Hence, it was proposed that the chaperone function of DnaK is
terpart revealed that the DnaK-dependent DNA binding activity
of HspR can indeed be stimulated in the presence of ATP, and
hence, the chaperone function of DnaK may have a role to play.
Although it is generally agreed upon that DnaK plays an im-
the mechanism by which it does so is not clearly understood. A
major problem regarding the study of HspR is that DnaK copuri-
fies with the native protein (2), and therefore, it is difficult to
feedback regulatory process. Purified preparations can be ob-
tained if HspR is isolated under denaturing conditions, but its
renaturation becomes a problem, as the protein is aggregation
prone. In this study, by isolating the M. tuberculosis HspR in the
denatured state and renaturing it using a gel filtration-based
free from any DnaK contamination. With the help of this rena-
DNA binding activity is due primarily to the presence of a hydro-
version of HspR which no longer requires DnaK for its activity.
MATERIALS AND METHODS
Bacterial strains and plasmids. Mycobacterial DnaK, N-terminally
tagged with 6? His residues, was purified after expression from plas-
& Vaccine Testing, NIH, NIAID, based at Colorado State University).
The M. tuberculosis HspR expression plasmid, pTDR30, was con-
structed earlier (8).
Chemicals. Nickel-nitrilotriacetic acid (Ni2?-NTA) agarose was pur-
chased from Qiagen (Valencia). For microtiter plate-based protein-pro-
tein interaction assays, Ni-NTA HisSorb plates (Qiagen, Valencia) were
used. Other chemicals for protein expression, purification, and analysis
were of the highest purity grade, obtained from SRL, India. [?-32P]ATP
(12 ? 1013Bq mmol?1) was purchased from BRIT (Mumbai, India).
alignments of multiple sequences were performed using the software
MEGA 4.0 (27). Multiple-alignment penalties of 10 and 0.1 were used for
chosen were either PAM or BLOSUM (10). The alignments created with
ment editor for convenient schematic representation. Phylogenetic trees
ysis was performed using 500 replicates.
Site-directed mutagenesis. To create a C-terminally truncated ver-
in the expression vector pTDR30 at position 119 of HspR using the
AAA CCG-3= and 5=-CGG TTT CCA GAC GAC CTA GGC GGT GCT
CTT CGG-3= (the mutation is underlined). The mutation was confirmed
by DNA sequencing. The resulting mutant plasmid was designated
Purification of recombinant proteins. Recombinant N-terminal 6?
His-tagged proteins were isolated by Ni2?-NTA agarose affinity chroma-
tography under either native or denaturing conditions using standard
protocols given by the suppliers of the affinity column (Qiagen) and as
described earlier (8). For DnaK-HspR interaction studies, an expression
construct from which an untagged version of DnaK could be synthesized
was made. The M. tuberculosis DnaK coding region (without the 6? His
codons) was amplified from the plasmid pMRLB.6 dnak/RV0350 using
the primers 5=-TTCGGATCCATATGGCTCGTGCGGTC-3= (forward)
and 5=-CCCCAAGCTT TCACTTGGCCTCCCG-3= (reverse) (NdeI and
HindIII sites underlined, respectively) and cloned into the multicloning
site of vector pT7-7 (26). The recombinant protein was isolated by am-
monium sulfate fractionation and ion-exchange chromatography essen-
tially as described earlier (13). The purity was more than 95%, as evident
from an SDS-PAGE analysis of the eluted protein.
urea was renatured by a stepwise reduction of the urea concentration by
fuged at 11,400 ? g for 15 min to remove aggregated proteins if any.
Removal of urea was also done by size exclusion chromatography (SEC).
One milliliter HspR (1 mg/ml) in buffer containing 8 M urea was layered
on top of a Sephacryl S-200 size exclusion column (bed volume, 40 ml)
equilibrated in buffer containing Tris-HCl (50 mM), KCl (200 mM),
EDTA (5 mM), MgCl2(5 mM), and 10% (vol/vol) glycerol. The column
was run at a flow rate of 0.50 ml/min. The fractions were then analyzed
using UV absorption spectrometry and SDS-PAGE analysis.
For rapid refolding in the presence or absence of chaperones, HspR
?l by dilution into renaturation buffer containing 20 mM Tris-HCl, pH
ation was done for 1 h at room temperature. In the case of DnaK-assisted
time period, the refolding mixture was centrifuged and the supernatant
was taken out. From this supernatant, aliquots (25 ?l) were removed and
used for electrophoretic mobility shift assays (EMSA).
Electrophoretic mobility shift assay. An electrophoretic mobility
shift assay was performed using a32P-labeled, 110-bp PCR-amplified
DNA fragment derived from the dnaKJE operon promoter-operator re-
gion (?128 to ?38) encompassing the two inverted HAIR repeats (25).
The primers used (K-128F and K-38R) and the protocols for performing
EMSA and antibody supershift assays were the same as those reported in
molar excess of either self unlabeled probe or an unrelated unlabeled
probe of similar size derived from the plasmid pAL5000 origin (1) was
incorporated in the preincubation phase.
Circular dichroism. Circular dichroism (CD) measurements were
done on a Jasco-815 spectropolarimeter using a 1-mm-pathway quartz
cell at 25°C. The far-UV CD spectra were recorded in the range of 200 to
scans were accumulated for each spectrum. The concentration of HspR
was 5 ?M.
Promoter assays. The dnaKJE-hspR promoter-operator region used
for EMSA was cloned in the lacZ-based promoter probe vector pSD5B
(12) at the XbaI site, resulting in the promoter construct pSDTD2. IPTG
ther the wild type (WT) (pTDR30) or the C-terminally truncated mutant
DnaK-Independent Renaturation of HspR
September 2012 Volume 194 Number 17jb.asm.org 4689
(?C) (pTDR30-1) hspR were cotransformed into E. coli along with
pSDTD2. In control experiments, pSD5S30B (9), a pSD5B-based con-
struct in which lacZ is expressed from S30, a randomly isolated Mycobac-
(7), was used in place of pSDTD2. Cotransformed cells were grown to
were either left alone or heat shocked at 42°C for 10 min. Recovery was
allowed for 40 min at 37°C, after which ?-galactosidase assays were per-
formed using the fluorescent substrate 4-methylumbelliferyl-?-D-galacto
pyranoside (MUG). Fluorescence readings were taken in the microtiter
plate reader POLARstar Optima (BMG Labtech). The results were ex-
pressed as arbitrary units of ?-galactosidase activity (MUG units), calcu-
lated using the equation (30) F/t ? A, where F, t, and A are sample fluo-
rescence intensity (excitation and emission at 360 and 460 nm,
respectively) at the end of the reaction, time of the reaction in minutes,
and absorbance of the cell suspension measured at 620 nm, respectively.
bit using the affinity-purified proteins DnaK and HspR. Preimmune and
Protein-protein interaction studies. The interaction between DnaK
and HspR was assessed by an enzyme-linked immunosorbent assay
(ELISA)-based method, which was used earlier to study the interaction
between DnaK and a plasmid replication protein (16), with some modi-
fications. Urea (8 M)-denatured 6? His-tagged HspR (75 ?M) was di-
luted 100 times in 100 ?l of renaturation buffer containing untagged
way that the final concentration reached 0.75 ?M, the same as in the case
of the denatured protein. As a negative control, a mock-interaction assay
up by omitting both. Incubation was done for 1 h at 25°C. The reaction
mixtures were split into two halves and added to the wells of a Ni-NTA
microtiter plate (Ni-NTA HisSorb). One half was processed for detecting
amount of HspR immobilized. Following adsorption, the wells were
blocked with phosphate-buffered saline containing 0.2% bovine serum
albumin (PBS-BSA) for 1 h under mild shaking conditions, followed by
several washings with PBS containing 0.05% Tween 20. One well of each
duplicate set was probed with anti-HspR sera, and the other was probed
with anti-DnaK (1:250 dilutions). After washing and addition of alkaline
phosphatase-conjugated secondary antibody, color development was
done using the chromogenic substrate para-nitrophenyl phosphate
Color development was monitored by measuring the absorbance at 405
nm (A405) using a microtiter plate reader. An A405corresponding to the
reagent blank was considered to be zero.
ment of HspR sequences from various Eubacteria using ClustalW
revealed that the N-terminal half of the protein, which includes
the winged helix-turn-helix motif DNA binding domain (HTH-
W1-W2), is highly conserved (Fig. 1A). In contrast, the sequence
in the C-terminal region is variable. Phylogenetic analysis based
a distinct and possibly ancient clade. These HspRs possess a du-
plication of their HspR domains, designated 1 and 2. HspRs of
more-modern Eubacteria, the Campylobacterales, Campylobacter
these HspRs appear to be closely related to the HspR of Aquifex
aeolicus, a hyperthermophilic bacterium belonging to the ancient
terales may have acquired their hspR orthologs from ancient ther-
mophilic bacteria through horizontal transfer. The third clade
comprises HspRs derived from bacteria belonging to the phylum
Actinobacteria. Within this clade, a subgroup of HspRs derived
from Actinomycetales form a distinct branch. The HspRs of this
group are characterized by the presence of a C-terminally located
conserved motif comprising the hydrophobic sequence LVVW
flanked by the positively charged amino acid residues lysine and
arginine (Fig. 1A, boxed region). HspR of Propionibacterium is,
however, an exception, in that although it is derived from an ac-
tinomycete, it clusters with the bifidobacterial HspR (Fig. 1B). It
has been established in earlier studies that DnaK preferentially
positively charged residues in the flanking regions increase bind-
ing (18, 19). The LVVW core is hydrophobic and also flanked by
positively charged amino acid residues. Hence, it was hypothe-
sized that this motif may be involved in the interaction of HspR
with DnaK. To test this hypothesis, a C-terminal deletion mutant
(HspR-?C) was constructed to exclude the LVVW motif.
Renaturation of HspR-WT and HspR-?C. The LVVW se-
quence is highly hydrophobic and includes a tryptophanyl resi-
due, considered to be the most hydrophobic of all amino acids
ied (6). However, if they are in the exposed state, they may trigger
aggregation. It was suspected that the LVVW motif may be re-
to form aggregates, as reported earlier (2, 8). To test this possibil-
ity, attempts were made to renature HspR-WT and HspR-?C
from the denatured state by stepwise removal of the denaturant,
and 5 compared to lane 1). HspR-?C, on the other hand, re-
mained soluble. Most of the protein (?87.5%) was recovered in
the soluble supernatant, whereas a small amount (?12.5%) was
found to be present in the pellet fraction (Fig. 2A, lanes 4 and 6
compared to lane 2). Since HspR-WT could not be renatured
efficiently through dialysis, an alternative strategy based on SEC
(31) was attempted. HspR isolated under denaturing conditions
through affinity purification was loaded onto a SEC column (ex-
protein eluted as a single peak corresponding to the HspR mono-
mer (Fig. 2B and C). The secondary structure of HspR present in
From the spectra (Fig. 2D and E), the helical contents of both of
the renatured proteins were calculated to be about 33%. The re-
HspR-?C) leads to significant improvement in its solubility and
ability to refold spontaneously. The results also indicate that the
WT protein can be renatured effectively by performing SEC.
DNA binding activity of renatured HspR. DNA binding as-
says were performed using HspR-WT and HspR-?C, renatured
by SEC (WTSE) and dialysis (?CD), respectively. The probe used
was a32P-labeled, 110-bp DNA segment encompassing the HspR
Bandyopadhyay et al.
jb.asm.org Journal of Bacteriology
from a broad spectrum of Eubacteria using MEGA 4.0. The similar/identical residues (PAM matrix) were colored using BioEdit tools. The motif conserved in
the associated wing domains (W1 and W2) are indicated on the top. The abbreviations are as follows. Mtb, Mycobacterium tuberculosis; Sco, Streptomyces
Bifidobacterium breve; Cje, Campylobacter jejuni; Hpy, Helicobacter pylori; Aae, Aquifex aeolicus; Dra, Deinococcus radiodurans; Dge, Deinococcus geothermalis;
between 17 HspR taxa. The conserved winged HTH (HTH-W1-W2) region of the alignment (A) was used for tree construction using the neighbor-joining
DnaK-Independent Renaturation of HspR
September 2012 Volume 194 Number 17jb.asm.org 4691
operon (?128 to ?38). A concentration-dependent increase in
the lower concentration, C1 is formed, but as the concentration
idea about specificities, competition binding experiments were
performed using a molar excess of either self (specific) and or an
unrelated (nonspecific) competitor, a 150-bp fragment derived
(Fig. 3B and C). The resulting competition experiments revealed
ner by only the specific and not the nonspecific competitor. This
indicates that the complexes were specific. Moreover, the rates of
competition by the self competitor were almost the same for WT
site did not change significantly following removal of the C-ter-
out DnaK assistance. In order to test whether DnaK assistance is
necessary under heat shock conditions, EMSA experiments were
FIG 2 Renaturation efficiencies of HspR-WT and HspR-?C. (A) Renaturation by dialysis. A 500-?l aliquot of the urea-denatured sample was dialyzed serially
against buffers containing decreasing concentrations of urea. After complete removal of urea, the dialyzed sample was centrifuged to separate the soluble
taken from the supernatants and subjected to SDS-PAGE analysis (lanes 3 and 4). For comparison, an identical volume taken from the sample prior to dialysis
SEC. The A280(absorbance at 280 nm) profile of the eluted protein (B) is shown along with the SDS-PAGE analysis (C) of fractions under the major peak. The
the serial dialysis methods, respectively.
in a final reaction volume of 30 ?l. The complexes are marked as C1 and C2. (B and C) Competition binding assays performed with WT and ?C proteins,
Bandyopadhyay et al.
jb.asm.org Journal of Bacteriology
performed using the heat shock proteins, either WT or ?C, in the
presence or absence of either DnaK or Hsp16.3, a chaperone
results show that in the absence of heat shock, HspR formed the
specific complexes C1 and C2 (Fig. 4, lane 2). In the presence of
a low-intensity band (C3) in the presence of DnaK (Fig. 4, lane 3)
ished (Fig. 4, lane 5). Upon addition of DnaK, the DNA binding
activity of HspR was restored, as indicated by the reappearance of
the major HspR DNA complexes C1 and C2 (Fig. 4, lane 6). The
minor complex C3 was also formed in addition to C1 and C2.
ble of preventing loss of HspR’s DNA binding activity. Neither
DnaK nor Hsp16.3 possesses any DNA binding activities of its
to direct binding of these chaperones to the probe. The results
obtained suggest that the presence of DnaK or, to a lesser extent,
any other chaperone can assist HspR by preventing its denatur-
ation under heat shock conditions. In sharp contrast, HspR-?C
Hsp16.3 made no difference (Fig. 4, lanes 8 to 13). These results
clearly indicate that removal of the C-terminal tail leads to stabi-
lization of the protein on one hand and DnaK independence on
Renaturation of urea-denatured HspR. While heat shock at
either completely or substantially denature it. The question that
was raised was whether DnaK can help in the renaturation of
HspR once it has been completely denatured. The ability of HspR
to renature to the active form, either spontaneously or with the
assistance of DnaK, was functionally investigated by performing
EMSA experiments with the denatured samples that were rena-
tured by dilution into denaturant-free buffer in the presence or
absence of DnaK.
The results show that when denatured HspR-WT was diluted,
no binding activity could be recovered, indicating that HspR-WT
was unable to regain activity (Fig. 5A, lane 2). On the other hand,
when denatured HspR-?C was similarly diluted, formation of
complexes C1 and C2 was observed (Fig. 5A, lane 4). This indi-
cated that HspR-?C, but not HspR-WT, could be renatured by
dilution into denaturant-free buffer without the assistance of
DnaK. When denatured HspR-WT was diluted into DnaK con-
taining renaturation buffer, DNA binding activity was observed,
lane 3). Unlike HspR-WT, denatured HspR-?C did not form C4
in the presence of DnaK. The predominant complexes were C1
and C2 (Fig. 5, lane 5). However, the minor complex C3 was also
found to be present.
To test whether the C4 complex was specific, competition
itors. The results showed that a 750-fold molar excess of the spe-
cific competitor abolished complex formation, whereas at the
same molar excess, the nonspecific competitor failed to compete
(Fig. 5B). This indicates that the complex C4, like C1 and C2, is
DnaK, it was necessary to examine whether the added DnaK be-
came a part of complex C4. A supershift assay was performed
using antisera against DnaK. The corresponding preimmune sera
were used as a control. The results show that in the presence of
anti-DnaK sera, a supershift was observed (Fig. 5C). The preim-
mune sera had no effect. This indicates that DnaK is a part of
complex C4. The important conclusions from these experiments
are as follows: (i) the C-terminal tail of HspR plays a key role in
DnaK-HspR interactions. In order to assess the affinity of
DnaK for HspR-WT and HspR-?C, direct binding assays were
6? His-tagged HspR, either WT or ?C, was allowed to bind in
solution to a tag-free version of mycobacterial DnaK. The
DnaK-HspR complexes formed were immobilized onto the
wells of a Ni-NTA HisSorb plate in duplicate. One well of each
duplicate set was probed with anti-HspR sera, and the other
was probed with anti-DnaK. The results show that DnaK bind-
ing was the maximum in the case of denatured HspR-WT (Fig.
6A). In the case of renatured HspR-WT, binding was only mar-
ginally higher than background levels. That this difference is
not due to variations in the amount of HspR present in the
assay system is evident from the observation that there were
only minor differences in the corresponding A405(HspR) val-
ues (Fig. 6B). As in the case of the renatured WT protein, the
level of DnaK binding to renatured HspR-?C was low, but the
interesting observation was that unlike in the case of the WT,
denatured HspR-?C did not show any significant binding to
DnaK. The observed differences became more pronounced if
the minor fluctuations in the amount of HspR bound to the
support were taken into account and the data were normalized
FIG 4 DNA binding activity of HspR under heat shock conditions. EMSA
experiments were performed using the WTSEor ?CDprotein (1 ?M) exposed
to either room (25°C) or heat shock (42°C) temperature in the presence or
absence of either DnaK or Hsp16.3 (2 ?M). In the case of the DnaK reactions,
of the free probe. The complexes C1, C2, and C3 are indicated.
DnaK-Independent Renaturation of HspR
September 2012 Volume 194 Number 17 jb.asm.org 4693
accordingly (Fig. 6C). Such normalization was done by finding
the ratios (absorbance ratios) between the respective A405val-
ues for DnaK and HspR. The major conclusions from this ex-
periment are as follows. (i) The hydrophobic C-terminal tail
plays an important role in the ability of HspR to interact with
the DnaK. (ii) Presence of the tail is not enough; it has to be
present in the exposed (denatured) state for productive inter-
action with DnaK.
In vivo implications of deleting the C-terminal tail. In order
to test whether deletion of the C-terminal tail affects the ability
the surrogate host E. coli. Reporter activity from pSDTD2 or
the control plasmid pSD5S30B, which expresses the reporter
gene lacZ from the unrelated S30 promoter, not known to be
heat inducible, was assessed in the absence or presence of HspR
synthesized from the cotransformed plasmids pTDR30 (for
HspR-WT) and pTDR30-1 (for HspR-?C). Following induc-
tion of HspR synthesis, the samples were divided into two
parts; one part received no heat shock, while the other did (Fig.
7A and B, respectively). The results showed that the reporter
activities from both pSDTD2 and pSD5S30B were higher than
that of the promoterless vector pSD5B. Hence, both promoters
were active in E. coli. The activity of pSDTD2 was, however,
(pSDTD2?WT), partial repression of lacZ expression was ob-
served in the absence of heat shock (Fig. 7A), but for HspR-?C
(pSDTD2??C), repression was near complete. Upon heat
shock (Fig. 7B), derepression from the dnaKJE-hspR promoter
was clearly evident in the case in which repression was medi-
ated by the WT repressor (pSDTD2?WT), but no such effect
was observed in the case of the ?C mutant (pSDTD2??C).
the case of the WT, heat shock could not relieve the repression
mediated by HspR-?C. These effects are clearly specific to the
dnaKJE-hspR operon promoter, as expression from the control
buffer containing 0.5 mM ATP in the presence or absence of 2 ?M DnaK. Following preincubation, labeled probe was added. (B) A competitive EMSA was
performed to examine the specificity of the C4 complex formed in the case of HspR-WT, using the molar excess of self (specific) or unrelated (nonspecific)
panels B and C contain no protein. The position of the band corresponding to the free probe (F) is shown in each autoradiogram.
FIG 6 ELISA to study the interaction between DnaK and either WT or ?C HspR. Mean A405values derived from five replicate ELISAs using antisera against
in which HspR was missing.
Bandyopadhyay et al.
jb.asm.org Journal of Bacteriology
of either WT or ?C HspR. Small differences, if any, were found
to be statistically insignificant.
Mycobacterium tuberculosis HspR is a repressor that controls the
onstrated earlier that as in the case of Streptomyces coelicolor (2),
the activity of M. tuberculosis HspR is modulated by DnaK (8).
HspRs from Actinomycetales, such as M. tuberculosis and S. coeli-
present (2, 8). A sequence comparison of HspRs from diverse
groups of bacteria revealed that although HspR is a highly con-
served protein, there are significant sequence variations in the
C-terminal region. Phylogenetic analysis revealed that HspRs can
be divided into two broad groups. One group comprises the
HspRs from ancient thermophilic bacteria, whereas the other
group comprises the Actinomycetales HspRs. Surprisingly, the
those derived from thermophilic bacteria. In particular, their
HspRs were found to be closely related to that of the ancient hy-
perthermophile Aquifex aeolicus. It appears that the Campylobac-
terales may have acquired their hspRs from ancient bacteria
through horizontal transfer.
The actinobacterial HspRs form a distinct clade, and most of
them possess the conserved hydrophobic motif LVVW in the C-
terminal region which potentially constitutes a high-affinity
DnaK binding site (18). To investigate the role of this motif in
HspR’s DnaK-dependent DNA binding activity, the C-terminal
tail region of the M. tuberculosis HspR was deleted so as to elimi-
nate this motif. The solubility of the mutant protein (HspR-?C)
easily renatured by dialysis against denaturant-free buffer or by
simple dilution into renaturation buffer. The amount of protein
the WT. As HspR-WT could not be renatured by these methods,
an alternative strategy in which removal of the denaturant was
done using size exclusion chromatography was used (31). This
denaturant gets removed in infinitesimal steps and, therefore, the
protein gets more opportunity to achieve the biologically active
EMSA experiments with the refolded proteins revealed that
both HspR-WT and HspR-?C were capable of binding to the
target site unaided. The binding patterns were the same for both.
Two complexes, C1 and C2, were formed in a concentration-de-
at the higher, C2 was formed. C2 migrated more slowly than C1,
operon operator region (25), it is most likely that C1 is mono-
meric and C2 is dimeric. The binding was found to be specific, as
the complexes formed were competed out by the self competitor
but not by an unrelated DNA fragment. Marginal batch-to-batch
differences in binding efficiencies were encountered, probably
due to differences in renaturation efficiencies. This might explain
why, in the concentration-dependent DNA binding experiment,
the observed binding efficiency of HspR-?C was somewhat less
than that of the WT. The proportion of active molecules in the
HspR-?C preparation used in this experiment was probably less
than that of the WT. An alternative possibility is that HspR-?C
competition DNA binding assays, however, do not support this.
In both cases, increasing concentrations of the self competitor
abolished complex formation at almost the same rate. The affini-
ties of the two proteins for the target site are thus unlikely to be
independent of DnaK, resulting in the complexes C1 and C2, and
atorily required (2). However, DnaK certainly has a role to play,
ature of 42°C. It is in this context that the presence of DnaK be-
comes relevant. It protects against thermal denaturation of rena-
tured HspR. At a lower level, the chaperone Hsp16.3, known for
its ability to prevent thermal denaturation of proteins (4), also
gave protection. This indicates that as far as protection against
FIG 7 In vivo effect of C-terminal truncation. Fluorescence-based ? galacto-
sidase reporter assays of E. coli cells transformed with the indicated promoter
experimental sets without or with heat shock, respectively. The results are
expressed in MUG units (means ? standard deviations for experiments per-
formed independently five times). For determining the significance of the
differences between group means, unpaired t tests were performed between
the control group (pSDTD2 or pSD5S30B) and the experimental groups (ei-
0.05; ****, P ? 0.0001).
DnaK-Independent Renaturation of HspR
September 2012 Volume 194 Number 17jb.asm.org 4695
partially substitute for DnaK. The introduction of DnaK did not
result in further retardation in the movement of C1 and C2.
Hence, the protective action does not appear to involve complex
formation between HspR and DnaK. In the presence of DnaK,
complex is not clear at present. No evidence for the presence of
DnaK was obtained using antibody supershift assays (data not
shown). It is likely to represent a complex involving HspR multi-
mers. Even if it is assumed that DnaK is a part of C3, the basic
conclusion that DnaK gives protection against the thermal insta-
bility of HspR without forming a complex remains unchanged, as
the mobilities of the DnaK-independent complexes C1 and C2
remain unaffected. It has been demonstrated in this study that
DnaK has high affinity for wild-type protein, particularly when it
interacted with DnaK, resulting in a C3-type complex.
Unlike protection, renaturation of the denatured protein ob-
ligatorily requires formation of a stable complex between DnaK
and HspR. Under this condition, a single retarded complex (C4)
which migrates more slowly than all the other complexes is
formed. Supershift assays convincingly demonstrate that DnaK is
a part of the complex. The results of protein-protein interaction
tured state, then the formation of a DnaK-HspR complex is facil-
C4 did not form and the DnaK-independent complexes C1 and
C2 were the predominant species. This indicates that DnaK did
tured state. This observation finds support from the results of the
protein-protein interaction experiment, which shows that, in-
deed, HspR-?C has little or no DnaK binding activity, even when
it is present in the denatured state. The results thus obtained
clearly indicate that the C-terminal hydrophobic tail of HspR
which includes the LVVW motif is the primary site where DnaK
binds. However, considering that only the denatured form of the
WT protein gave a high level of DnaK binding activity, it may be
and that it must be in the exposed state so that interaction with
DnaK is possible. A minor complex of the C3 type was neverthe-
less seen in the case of denatured HspR-?C. Hence, although the
LVVW motif constitutes the primary DnaK binding site, there
may be others which are secondary in nature.
To understand how the C-terminal deletion affects the repres-
coli as a surrogate host. E. coli does not have an ortholog of HspR.
Hence, there is no possibility of background interference. The
ability of HspR to repress the activity of the dnaKJE-hspR operon
promoter in trans was demonstrated using reporter assays. Dere-
pression of the operon promoter was found to occur under heat
shock conditions. HspR-WT, however, appeared to be a weak re-
pressor, as it repressed only partially. In contrast, HspR-?C
promoter to the level observed in the case of the promoterless
was observed in the case of HspR-?C. This observation gives a
valuable clue regarding the physiological significance of the pres-
ence of the C-terminal hydrophobic motif. It is due to the pres-
ence of this motif that the protein becomes unstable and, there-
fore, the system responds to heat efficiently. It is interesting that
although HspR-WT and HspR-?C were demonstrated to have
nearly identical DNA binding affinities in vitro, in vivo, HspR-?C
was found to be more efficient. This can be explained on the basis
of the differences in the thermal stabilities of the WT and mutant
proteins. While the in vitro DNA binding experiments were all
performed at 0°C, the in vivo experiments required that the tem-
perature be maintained at 37°C so as to allow optimal bacterial
growth. The WT protein, being thermally unstable, may be only
partially active at the relatively high temperature of 37°C, and
therefore, repression was leaky. In the case of HspR-?C, which is
thermally stable, there is no such constraint, and hence, it func-
tions as a better repressor.
HspRs can be divided into two types depending on their solu-
bility—the relatively insoluble type found in the Actinomycetales
(2, 8, 29) and the more-soluble types found in Campylobacterales
(23) and bacteria belonging to the phylum Thermus-Deinococcus
more dependent on feedback control is an intriguing question.
cases. Thus, the C-terminal tail of chlamydial HrcA was found to
inhibit its DNA binding activity, and in this case, GroEL can re-
lieve this inhibition, apparently by interacting with the tail (5).
Negative regulation of heat shock operon repressors by their own
C-terminal tails may constitute a universal strategy by which Eu-
bacteria control their responses to heat shock.
We thank CSIR and DST (Government of India) for financial support.
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