Functionalsimilaritiesbetweenthesmallheatshock proteins
Mycobacterium tuberculosis
HSP 16.3andhuman aB-crystallin
Melissa M. Valdez
1
, John I. Clark
1,2
, Gabrielle J. S. Wu
3
and Paul J. Muchowski
4
1
Departments of Biological Structure, and
2
Ophthalmology, University of Washington, Seattle, WA, USA;
3
Seattle Genetics, Bothell,
WA, USA;
4
Department of Pharmacology, University of Washington, Seattle, WA, USA
Mycobacterium tuberculosisheatshock protein 16.3 (MTB
HSP 16.3) accumulates as the dominant protein in the latent
stationary phase of tuberculosis infection. MTB HSP 16.3
displays several characteristics of smallheatshock proteins
(sHsps): its expression is increased in response to stress, it
protects against protein a ggregation in v itro, and it contains
the core Ôa-crystallinÕ domain found in all sHsps. In this study
we characterized the c haperone activity of r ecombinant
MTB HSP16.3 in several different assays and compared the
results to those obtained with recombinant human
aB-crystallin, a well characterized member of the sHsp
family. Recombinant MTB HSP16.3 w as expressed in
Escherich ia coli and purified to apparent homogeneity.
Similar to aB-crystallin, MTB HSP16.3 suppressed citrate
synthase aggregation and in the presence of 3.5 m
M
ATP the
chaperone activity was e nhanced by twofold. ATP stabilized
MTB HSP16.3 against proteolysis by chymotrypsin, and no
effect was o bserved w ith ATP cS, a nonhydrolyzable analog
of ATP. Increased expression of MTB H SP 16.3 resulted in
protection against thermal killing in E. co li at 48 °C. While
the sequence similarity betweenhumanaB-crystallin and
MTB HSP16.3 is only 18%, these results suggest that the
functional similaritiesbetween t hese proteins containing the
core Ôa-crystallinÕ domain are much closer.
Keywords: ATP; human aB-crystallin; molecular chaperone;
Mycobacterium tuberculosisHSP16.3 ; smallheat s hock
proteins.
One-third of the world’s population is infected with latent
inactive tuberculosisand active tuberculosis is the leading
cause of d eath due to an infectious disease [ 1]. Each year,
new infections occur in 54 million people; 6.8 million people
develop c linical disease, and 2 .4 million cases result in death
[2]. There is still limited knowledge of the molecular
pathogenesis of the latent stage of this organism [3].
Individuals w ho have been infected with Mycobacterium
tuberculosis can harbor stable dormant bacilli for decades
before developing an active infection later in life [4]. Recent
reports indicate an important role for M. tuberculosis
(MTB) heatshock protein (HSP) 16.3 in the survival of
MTB during p rolonged periods of infection [5–7]. It w as
shown that MTB HSP 16.3, initially described as the
immunodominant 14- or 16-kDa antigen [8–11], was a
major component in tuberculosis infection in humans and
played an important role in enhancing protein stability and
survival [5]. Eighty-five percent of patients with active
tuberculosis showed a positive reaction to this a ntigen,
suggesting that this protein expressed in vivo had a key role
in MTB infection [11,12]. The 14K antigen was later
renamed MTB HSP16.3 [13]. MTB HSP 1 6.3 accumulates
to become t he dominant protein in the la tent stationary
phase of M. tube rculosis infection [7]. Over-expression of
HSP 16.3 in log phase growth of M. tuberculosis slowed the
growth rate and protected ag ainst stationary phase autolysis
in v itro [7]. MTB HSP16.3 h as been charac terized a s a
membrane associated protein [12] having sequence homo-
logy to other proteins i n thesmallheat s hock protein (sHsp)
family [11,14]. All sHsps share se quence similarity in a
conserved 80–100 amino-acid Ôa-crystallinÕ domain region
found in the C-terminus which is thought to be important
for c haperone function s [14–16]. MTB HSP16.3 h as been
shown to contain an oligomeric, active structure which may
form a trimer of trimers and pos sesses in vitro molecular
chaperone activity [13].
Up-regulation o f l arge andsmall sHsps is t hought to be a
universal response to s tress. In vitro, human aB-crystallin
and other sHsps function as molecular chaperones by
suppressing unfolding and aggregation o f polypeptides in
response to s tress [17,18]. MTB HSP16.3 modulates its
chaperone activity by exposing hydrophobic s urfaces a nd
demonstrates conformational flexibility allowing maximum
interactions with denaturing proteins [19]. A recent paper
reports that th e only universally con served leucine residue
among all the members of the sHsp family plays an
important role i n m olecular c haperone activity of MTB
HSP 16.3and oligomeric structure formation [20]. I t has
been reported t hat the chaperone activity of MTB H SP 16.3
is independent of the effects of ATP [13,19]. In contrast,
molecular chaperones of the large heatshock protein
families suppress protein unfolding and aggregation during
stress and participate in the refolding of denatured proteins
in vitro, in an ATP-dependent manner [21,22]. While the
molecular chaperone effects of the sHsp do not r equire
ATP, the activity of human- aB crystallin was enhanced with
Correspondence to J. I. Clark, Department of Biological Structure,
Box 357420, University of Washingto n, Seattle, W A 98195-7420,
USA. Fa x: +1 206 543 1524, Tel.: 1 206 685 0950,
E-mail: clarkji@u.washington.edu
Abbreviations: MTB HSP 16.3, Mycobacte rium Tuberculosis heat
shock protein 16.3; sHsps, smallheatshock proteins; IPTG, isopropyl
thio-b-
D
-galactoside; CFUs, colony forming units; CS, citrate
synthase.
(Received 12 July 2001, r evised 1 7 January 2 002, accepted 25 January
2002)
Eur. J. Biochem. 269, 1806–1813 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02812.x
ATP [23]. Recent reports confirmed the effect of ATP on
sHsps using bovine a-crystallin to stabilize partially dena-
turedproteinsduringreactivationinanATP-dependent
manner [24] and indicating the involvement of ATP in
substrate release [25]. In this s tudy, t he effect of ATP on
recombinant MTB HSP16.3 was compared with the effect
of ATP on recombinant human aB-crystallin.
We report t he expression and purification of recombinant
MTB HSP16.3 from E. coli and studies of its f unction as a
molecular chaperone. MTB HSP16.3 was compared with
aB-crystallin in vivo and in vitro biochemically and in
functional assays. Although only 18% sequence identity is
shared betweenthe two s Hsps, MTB HSP16.3 functioned as
effectively as aB-crystallin as a molecular chaperone in v itro.
The molecular chaperone activity of recombinant MTB
HSP 1 6.3 was enh anced in the presence of ATP which is
consistent with previous findings of the effect of ATP on
recombinant humanaB-crystallin [23]. The expression of
MTB HSP16.3 in E. coli exposed to high temperatures
resulted in a very impressive level of survival. Our results
suggest the chaperone activity ofMTB H SP 16.3may play an
important role in the survival and stability of M. tuberculo sis.
MATERIALS AND METHODS
Expression and purification of MTB HSP 16.3
HSP 1 6.3 was subcloned into the pET-20b(+) expression
vector which w as provided by H. McHaourab (Department
of Molecular Physiology a nd Biophysics, Vanderbilt
University School of Medicine, Nashville, TN, USA). T he
pET-20b(+)-HSP 16.3 expression plasmid was used to
transform E. coli BL21 (DE3) competent cells (Novagen,
Inc., Milwaukee, WI, USA). The expression of HSP 16.3
was based on a method described previously [26]. One litre
of Luria–Bertani broth containing 10 g NaCl, 5 g yeast
extract, and 10 g tryptone (DIFCO Laboratories), p H 7.0
with 50 lgÆmL
)1
carbenicillian was inoculated with 10 mL
of an overnight c ulture containing the pET-20b(+)-
HSP16.3 vector. The flasks were incubated for a total of
3 h at 37 °C until D
600
¼ 0.8–1.0. The cells were then
induced with 0.4 m
M
isopropyl thio-b-
D
-galactoside (IPTG)
for another 4 h. Cells were then harvested by sedimentation
and frozen at )20 °C until further use. Cell pellets of the
pET-20b(+)-HSP 16.3 were then lysed with 1 0 mL l ysis
buffer (20 m
M
Tris/HCl pH 7.0) and transferred to a small
beaker which was placed in ice. Forty microliters of 50 m
M
phenylmethanesulfonyl fluoride and 400 lL10mgÆmL
)1
lysozyme were added with constant stirring for 10 min;
20 mg deoxycholic acid were then added with an additional
10 min of stirring. The m ixture was removed from the ice
bath and 200 lLof1mgÆmL
)1
DNAse was added with
stirring for 30 min. The sample was then placed in a 50-mL
tube and centrifuged at 18 000 g for 1 h. The supernatant
from this sample was transferred to a new beaker with
constant stirring at room temperature with the addition of
400 lL 5% polyethylenimine a nd 800 lL 200 m
M
dithio-
threitol for 10 min. The sample was then c e ntrifuged at
35 000 g for 2 h at 4 °C. The supernatant was decanted and
the insoluble pellet w as discarded. The s upernatant was then
ready for purification using the Pharmacia FPLC system.
The supernatant containing the soluble protein was
filtered through a 0 .22 lm filter and was loaded onto a High
Trap Q Anion Exchange Column (Pharmacia), pre-equil-
ibrated with Buffer A (20 m
M
Bis/Tris, pH 6.5). The
proteins were eluted using a linear gradient of
0–1.0
M
NaCl. T he protein fractions were analyzed using
SDS/PAGE (Invitrogen). Proteins were analyzed on a
4–12% polyacrylamide e lectrophoretic gel in the presence of
0.1% SDS a nd Mes buffer and were stained with C oomasie
blue R-350 (Amersham Pharmacia). Fractions containing
the 16.3-kDa p rotein were th en pooled and concentrated
using a 1 0 000 molecular mass cut-off concentrator
(Amicon). Concentrated protein (5 mL) was loaded onto
a Phenyl Superose Hydrophobic Interaction Column,
preequilibrated with a buffer containing 50 m
M
sodium
phosphate, and 1.0
M
ammonium sulfate, pH 7.0. The
protein was then eluted using 50 m
M
sodium phosphate,
pH 7.0. The protein fractions were analyzed by SDS/PAGE
and all fractions containing the protein were pooled and
concentrated to 500 lL. The concentrated sample was
applied to a Superdex 200 HR 10/30 size exclusion column
(Pharmacia) and purified by gel filtration. Fractions
containing the p rotein w ere collected, c oncentrated, a nd
then q uantified for total protein concentration u sing the
Bradford method (Bio-Rad).
To confirm protein purification of HSP 16.3, a Western
immunoblot analysis was performed using monoclonal
antibody IT-4 (a-16 kDa) provided by D. Sherman
(Department of Pathobiology, University of Washington,
Seattle, WA, USA). Detection of protein was perfo rmed
using NEN Western Blot Chemiluminescence Reagents
(NEN Life Science P roducts Inc.). The s eq uence of t he
purified recombinant HSP16.3 was confirmed b y
MS. Automatic Edman sequencing was used with an applied
Biosystems model 470 A automatic protein sequencer.
In vivo
cell viability experiment with HSP16.3 at 48 °C
A XbaI–XhoI fragment containing the entire coding
sequence of HSP16.3 was excised from pET20b(+)-
HSP 1 6.3 and ligated into pET16b (Pharmacia) digested
with the same restriction enzymes. Restriction mapping
analysis was performed on pET16b-HSP 16.3 to ensure
that theHSP16.3 gene was i nserted in the proper
orientation, andthe coding sequence of HSP16.3 was
verified by DNA sequence analysis. The pET16b-HSP 16.3
vector was then t ransformed into BL21 (DE3) competent
cells.
For t he thermal killing experiment, an equal number of
cells were gr own containing either pET16b-HSP 16.3
vector, pET16b (empty vector control), or pET16b-aB
(positive control). Equal numbers of cells from overnight
cultures were inoculated into 50 mL of
L
-broth medium
containing 100 lgÆlL
)1
carbenicillin and grown at 37 °C
until they reached an D
600
¼ 0.8. Protein expression was
then induced with 1.0 m
M
IPTG. After a 2 h i nduction,
samples were s hifted to a s haking water bath a t 4 8 °C.
Samples w ere r emoved at 3-h t ime points postinduction
and scored f or cell viability by p lating on Luria–Bertani
broth plates containing carbenicillin. Cell viability was
determined by counting the number of colony forming
units (CFUs) on each plate after heatshock at 48 °C
relative to the s tarting number of CFUs f ormed in each
culture p rior to heat shock. Total protein lysates from
cells that expressed HSP16.3and control cultures w ere
Ó FEBS 2002 Similaritiesbetween MTB HSP16.3andaB-crystallin (Eur. J. Biochem. 269) 1807
analyzed by SDS /PAGE as described above. The experi-
ment was repeated four times using duplicates of each cell
culture.
Chaperone assays
The t hermal unfolding and aggregation of citrate synthase
(CS; Roche Molecular Biochemicals) at 4 5 °Cwasdeter-
mined by measuring the absorption from light scattering at
320 n m in a Beckman Spectrophotometer over a period of
30 min. Native CS was diluted to a 15 l
M
working
solution containing 20 m
M
Tris/HCl (pH 7 .4), and
100 m
M
NaCl. To t est the molecular chaperone effect,
different molar ratios of HSP16.3 were diluted into a
reaction buffer containing 100 m
M
Tris/HCl (pH 7.4),
100 m
M
NaCl in the presence or absence of ATP (final
volume 400 lL). For testing the effects of A TP and the
nonhydrolyzable ATP analog ATPcS, the r eaction buffer
was equilibrated w ith 3.5 m
M
ATP (or A TPcS), 3 .5 m
M
MgCl
2
and 1 0 m
M
KCl b efore addition of HSP16.3 o r
CS. The protection from thermal aggregation of CS at
45 °C w ith HSP16.3 was a lso c ompared with t he same
molar ratios of aB-crystallin (predicted from monomeric
molecular weights).
Chymotrypsin digestion of HSP 16.3
Chymotrypsin digestion with HSP16.3 was base d on the
methods used for GroEL andaB-crystallin [27,28]. In
summary, f or each reaction 70 lgMTBHSP16.3were
diluted into a final volume of 100 lL buffer c ontaining
100 m
M
Tris/HCl, pH 7.4, 3.5 m
M
MgCl
2
,10m
M
KCl,
and 0.01% Tween-20. For reactions with ATP or ATPcS,
a final concentration of 3.5 m
M
ATP or ATPcSwas
added to th e reaction mixture. To each sample at time
point 0, 0.17, 0.51 or 1.36 lg chymotrypsin was added
from a stock solution of 0.17 mgÆmL
)1
.Sampleswere
maintained at 37 °C f or the duration o f the experiment.
Immediately after chymotrypsin addition, 13.5 lLofthe
reaction mixture was removed and quenched with 1.5 lL
100 m
M
phenylmethanesulfonyl fluoride, and placed on
ice. At 5-min time points, 13.5-lL aliquots were r emoved
and treated identically to the zero time point sample.
Samples were analyzed by SDS/PAGE analysis as
described above.
RESULTS
Expression and purification of recombinant MTB
HSP 16.3 in
E. coli
Figure 1 is the SDS/PAGE and Western immunoblot
analyses of the expression and purification of MTB
HSP 16.3. Induction of protein expression with IPTG
resulted in the a ppearance of a protein band at
approximately 16.3 k Da (Fig. 1 A, lanes 2 and 3). The
expressed M TB HSP16.3 was purified by a combination
of anion e xchange, hydrophobic i nteraction and s ize
exclusion chromatography (lanes 4–6). Wester n immuno-
blot analysis of recombinant MTB HSP16.3 was
performed using a monoclonal antibody [IT-4
(a-16 kDa)] raised against native MTB HSP 1 6.3
(Fig. 1 B). IT-4 (a-16 kDa) recognized the recombinant
MTB HSP16.3 from E. coli and did not react with
recombinant aB-crystallin (Fig. 1 B). The purification
yield obtained from 1 L of Luria–Bertani broth culture
was between 10 and 3 0 mg o f MTB HSP 16.3.
Fig. 1. Expression and Purification of MTB HSP 16.3. (A) The
expression an d p urification of MTB HSP16.3 was a nalyz ed b y S DS/
PAGE using 4–12 % Bis/Tris polyacrylamide gels in the presence of
Mes buffer. Lanes 1 and 7, m olec ular mass markers; lane 2, expression
protein in E. coli cells not induced by IPTG; lane 3, protein expression
in E. coli after induction with IPTG; lane 4, following purification on
the High trap Q anion exchange c olumn; lane 5, MTB HSP 16.3
enriched after purification using a Phenyl Superose H ydro phobic
Interaction Column; lane 6, following Tris/HCl buffer exchange of
MTB HSP16.3 on a Superdex 200 Size Exclusion column (to remove
salt). A protein assay performed after desalting the sample showed that
the y ields varied between 10 and 30 mgÆL
)1
of MTB HSP16.3 cell
culture. (B) SDS/PAGE (left) and Western immunoblot (right) on a
4–12% polyacrylamide gel of recombinant MTB HSP16.3 and
recombinant h uman aB-crystallin. Lane 1, molecular mass markers;
lane 2, MTB HSP 16.3; lane 3, aB-crystallin. In the Western immu-
noblot, th e I T-4 antibody to MTB HSP16.3 d etected r ecom binant
MTB HSP16.3 in lane 2 only. No reactivity with anti-(MTB
HSP 16.3) I g was obser ved in lan e 3, which contained human
aB-crystallin (right s ide).
1808 M. M. Valdez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Amino-acid sequence determination of recombinant
MTB HSP 16.3
The s equence of the purified recombinant MTB HSP 16.3
was c onfirmed b y M S. T he 1 2 r esidues a t the N-terminus of
MTB HSP16.3 were determined to b e: Ala-Thr-Thr-Leu-
Pro-Val-Gly-Arg-His-Pro-Arg-Ser. The N-terminal Met
residue was not identified by p rotein sequencing, as
observed previously [12,13].
Protection of cell viability at 48 °Cin
E. coli
by MTB
HSP 16.3and aB-crystallin
To characterize the protective e ffect of MTB HSP 16.3
over-exp ression in vivo, a thermal k illing assay in E. coli was
used (Fig. 2A). The number of CFUs w ere counted in
cultures of E. coli with and without MTB H SP 16.3 and
aB-crystallin shifted to 48 °C. The proportion of viable cells
that survived heatshock at 48 °C was plotted at three time
points (t ¼ 0, 3 and 6 h a fter 48 °C heat shock). C ells that
expressed MTB HSP16.3 or aB-crystallin were resistant to
thermal killing a t 48 °C over t he 6-h time c ourse of the
experiment (Fig. 2A). After 3 hs, the viability of cells th at
contained the empty c ontrol vector decreased b y four orders
of magnitude, while the viability of cells that over-expressed
MTB HSP16.3 o r aB-crystallin decreased only slightly.
Within 6 h of heatshock a t 48 °C, no viable ce lls were
observed in cells containing theaB-crystallin vector that
were not induced for protein e xpression. The viability by
6 h , decreased dramatically in cells containing the pET 16b
control vector as measured by the CFUs. In contrast, the
viability o f cells that over-expressed MTB HSP 16.3
decreased by two orders of magnitude after the 6-h heat
shock. The protective effect of the induced aB-crystallin was
stronger than MTB HSP 16.3. SDS/PAGE analysis s howed
strong expression of MTB HSP16.3andaB-crystallin at
time points 0 , 3, and 6 h following heatshock at 48 °C
(Fig. 2 B,C).
In vitro
chaperone activity of MTB HSP 16.3
and aB-crystallin
In Fig. 3, we observed the effects of different concentrations
of MTB HSP16.3 in the presence and absence of ATP on
the aggregation of CS. In the absence of added MTB
HSP 1 6.3, aggregation o f CS increased af ter a short delay to
reach a maximum after approximately 25 min at 4 5 °C
Fig. 2. Cell viability of MTB HSP 16.3. The pET 16b-MTB HSP 16.3
vector, the control pET 16b-aB vector andthe pET 16b vector con-
taining no inserted gene were e xpressed at 37 °C and induced with
IPTG when the cell c ultures reached D
600
¼ 0.8. After induction fo r
2 h andheatshock to 48 °C, the cells were incubated for a further 6 h.
Samples were taken at concurrent and sequential time points beginning
at the time of heat shock, plated and CFU counted. The proportions of
viable cells expressing the pET 16b-MTB HSP16.3 vector andthe two
control vectors were plotted for 0 , 3 and 6 h following h eat s hock. At
48 °C, the proportion of surviving cells expressing pET 16b vector only
or aB-crystallin uninduced c ells was negligible and viability of cell
cultures decreased more than fourfold (A). By 6 h post heat shock, the
cultures that over-expressed MTB HSP16.3andtheaB-crystallin in-
duced c ells remained viable. Protein expression was analyzed b y SDS/
PAGE on a 4–12% polyacrylamide gel in the presence of 0.1% SDS
and Mes bu ffer (B,C). Lanes 1 a nd 6 are molecular mass markers.
Lanes 2–5 show protein expression i n cells containing the pET 16b
vector alo ne a t selected times f rom 0 to 6 h. Lane 2 is the pro tein
expression in cells containing the pET 16b vector alone not induced
with IPTG. Lanes 3–5 are the pET 16b vector at the zero, 3 and 6 h
time points after 2 h of induction and post heatshock at 48 °C. Lane 7
of (B) i s protein ex pression in cells containing t he pet 16b-MTB
HSP 16.3 vector not in duce d with IPTG. Lanes 8 –10 a re pro tein
expression for the pET 16b-MTB HSP16.3 vector at time point zero, 3
and 6 h after 2 h of induction and post heat shock. (C) SDS/PAGE of
cells containing aB-crystallin induced and not induced with IPTG.
Lane 2–5 show aB-cr ystallin uninduced at the 0, 3 and 6 h time points.
Lane 7 contains cell homogenates of aB-crystallin n ot induced. Lane s
8–10 contain aB-crystallin induced with IPT G at the 0 , 3 and 6 h time
points. The cells expressing high levels of MTB HSP16.3 or aB-
crystallin (lanes 8–10) survived in culture at 48 °C(A).
Ó FEBS 2002 Similaritiesbetween MTB HSP16.3andaB-crystallin (Eur. J. Biochem. 269) 1809
(Fig. 3 A). With increasing ratios of M TB HSP 1 6.3 to C S, a
concentration-dependent suppression of aggregation was
observed over the 30-min period (Fig. 3 A). Complete
protection against aggregation was observed at a ratio of
15 : 1 MTB HSP 16.3:CS (monomer : monomer). The
addition of 3.5 m
M
ATP enhanced the effect of MTB-
HSP 16.3 on CS aggregation by approximately twofold
(Fig. 3 B). ATPcS, a nonhydrolyzable analog of ATP, did
not enhance t he effect of MTB H SP 16.3 on CS aggregation
(Fig. 3 C). T he chaperone activity of MTB H SP 16.3 was
next compared to aB-crystallin at identical molar ratios
(Fig. 4). In ge neral, aB-crystallin was more effective as a
molecular chaperone than MTB HSP16.3 under the
conditions of these experiments (Fig. 4). Complete s uppres-
sion of CS aggregation by MTB HSP16.3 required a molar
ratio of 15 : 1, while aB-crystallin required a molar ratio of
5 : 1 for complete suppression of aggregation.
Chymotrypsin proteolysis of MTB HSP 16.3
in the absence and presence of ATP
MTB H SP 16.3 was digested with chymotrypsin in the
absence and presence of ATP at 42 °C (Fig. 5A–C).
Proteolysis of MTB HSP16.3 increased with chymotrypsin
concentration as expected (data not shown). Each individ-
ual lane is a sample of MTB HSP16.3 plus chymotrypsin
Fig. 4. Comparison of molecular chaperone activity between recombin-
ant MTB HSP16.3and recombinan t human aB-crystallin. The
molecular chaperone activity of MTB HSP16.3 o n CS aggregation
was compared to the effect of humanaB-crystallin on C S aggregation.
The aggregation of CS was mea sured in the presence o f d ifferent
concentrations of MT B HSP16.3 o r humanaB-crystallin after a
30-min period. The bar graphs measure the a ggregation of C S in
arbitrary units vs. t he ratios of MTB HSP 16.3/CS and h uman
aB-crystallin/CS. With increased ratios of the molecular chaperone
protein to CS, there was increased protection aga inst CS aggregation.
Recombinant humanaB-crystallin demonstrated better p rotection
against CS aggregation than MTB HSP 16.3. At the 15 : 1 molar ratio
of MTB HSP 16.3/CS, protection aga inst CS aggregation was almost
complete. Similar protection was observed at a ratio of 5 : 1 for human
aB-crystallin/CS.
Fig. 3. Molecular Chaperone Activity of MTB HSP 16.3. To test the
molecular chaperone activity of MTB HSP 16.3, a series of aggrega-
tion assays was perform ed using CS with and without ATP over a
30-min period. The aggregation of CS was measured with the addition
of different concentrations of MTB HSP16.3and in the presence or
absence of ATP and ATP analogs. (A) Aggregation o f CS is plotted in
arbitrary units against time in the presence of increasing ratios of MTB
HSP 16.3 to CS. With the increase of MTB HSP 16.3, there was an
increase of protection against t hermal aggregation of CS [d, CS alone;
j, HSP 16.3/CS (5 : 1); ,, H SP 16.3/CS (10 : 1); ., HSP 16.3/CS
(12 : 1); s, HSP 16.3/CS (15 : 1)]. (B) Aggregation of CS plotted in
arbitrary units against time in the absence a nd presence of ATP at two
different molar concentrations of MTB HSP 16.3. In the presence of
ATP, the molecular chaperone effect of MTB HSP16.3 was enhanced
for aggregation of CS and maximum suppression of aggregation was
observed at a molar ratio of 10 : 1 HSP16.3 : CS [d, CS a lone;
,, HSP16.3 : CS (5 : 1); j, HSP16.3 : CS (5 : 1) + ATP; s,
HSP16.3:CS (10:1); ., HSP16.3 : CS (10 : 1) + ATP]. (C)
Control for the effe ct of ATP on t he mole cular c haperone ac tivit y of
MTB HSP 16.3. When 3.5 m
M
MgCl
2
and 1 m
M
KCl were added to a
solution containing MTB H SP 16.3and CS no effect on chaperone
activity was o bserved. The results using A TPcSandMgCl
2
with KCl
suggest the im portan ce of hydrolysis of ATP for c haperone activity of
MTB HSP 16.3. [d,CSalone;,,HSP16.3:CS (10:1)+1m
M
KCl and 3.5 m
M
MgCl
2
; s, HSP16.3 : CS (10 : 1); .,HSP 16.3 : CS
(10 : 1) + 3.5 m
M
ATPcS; j, HSP16.3 : CS (10 : 1) + 3.5 m
M
ATP].
1810 M. M. Valdez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
taken at 5-min time intervals over a 30-min period. Nearly
all intact MTB HSP16.3 was degraded after 15 m in in the
absence of ATP (Fig. 5A). In the presence of 3.5 m
M
ATP,
MTB HSP16.3 was stabilized against proteolysis, and
intact MTB HSP16.3 could be detected even after 3 0 min
of proteolysis (Fig. 5B). The digestion pattern of MTB
HSP 1 6.3 was similar in the absence and presence of ATP,
where two major proteolytic fragments at M
r
8and
13 kDa were observed. The specificity of the effect of ATP
on stabilization of MTB HSP16.3 against p roteolysis was
confirmed with nonhydrolyzable ATP analog ATPcS,
which had no stabilizing e ffect against proteolysis (Fig. 5C).
DISCUSSION
Our results demonstrated similar functions for recombinant
MTB HSP16.3andhumanaB-crystallin as molecular
chaperones, although the sequence identity between MTB
HSP 1 6.3 andaB-crystallin is only 18% (Fig. 6). MTB
HSP 1 6.3 is a sHsp that contains the conserved c ore
Ôa-crystallinÕ domain shared by m embers of the sHsp family
[14–16]. M. tuberculosisHSP16.3 was expressed and puri-
fied from E. coli for the comparative characterization on t he
molecular chaperone activity in vivo and in vitro with human
aB-crystallin, the well characterized archetype of the sHsp
family of molecular chaperones [14,16,29].
In vivo, the protective e ffect of MTB HSP16.3 expression
on the survival of E. coli in a thermal killing assay at 48 °C
was impressive (Fig. 2A). At 48 °C, there was approxi-
mately two orders of magnitude difference between survi-
ving cells expressing MTB HSP16.3and c ontrols without
MTB HSP16.3 expression. The results for MTB HSP 16.3
are consistent with previous reports with other sHsps
[23,30,31]. The protective effect of aB crystallin on cell
survival was s tronger than MTB HSP 16.3. I n p revious
in vivo studies, o ver-expression of HSP16.3 at the end of
log-phase growth in M. tuberculosis resulted in an enhanced
resistance to autolysis [ 5]. Our results s howing a protective
effect of MTB HSP16.3 against thermal killing in E. coli
are consistent with previous studies on the importance o f
MTB HSP16.3 expression in M. tuberculosis [5–7].
Although the role of MTB H SP 16.3 is not completely
understood, these experiments suggest that MTB HSP 16.3
may provide protection against cell death in M. tuberc ulosis.
In an in vitro aggregation assay using CS as a target
protein, MTB HSP16.3 was effective as a chaperone,
although less effective than aB-crystallin in suppressing CS
aggregation. It is possible that additional cofactors found
only in M. tuberculosis cytosol could i ncrease the chaper one
activity of MTB HSP 16.3. It is also likely t hat the efficiency
of MTB HSP16.3 as a chaperone may b e improved using
target proteins that are native to M. tuberculosis.
The e ffects of ATP on the c haperone activity of MTB
HSP 1 6.3 were similar to aB-crystallin, a sHsp whose
Fig. 6. Sequence alignment of recombinant MTB HSP16.3 and
recombinan t hum an aB-crystallin. Amino-acid sequence a lignment
between recombinant MTB H SP 16.3and h uman aB-crystallin was
aligned using the
MULTALIN MULTIPLE SEQUENCE ALIGNMENT
program
(PBIL, Franc e) with the h elp of S. Yarfitz (Unive rsity o f Washington
Health Science s L ibrary, Seattle, WA, USA). Shading indicates
chemically identical and similar a mino-acids residues (
BOXSHADE
program from the European Molecular Biology Network). Residues
highlighted black indicate amino-acid residues that are chemically
identical a nd residu es h igh lighted gray indicate amino-acid residues
that are c hemically similar. Between MTB HSP16.3and human
aB-crystallin there was an 18 % sequence identity and a n overall 30%
shared sequence similarity betweenthe tw o proteins. The conserved
core a-crystallin domain observed in proteins of the sHsp spans resi-
dues of E67–I161 in the aB- crystallin sequence.
Fig. 5. The chymotrypsin proteolysis of MTB HSP 16.3. SDS/PAGE used 4–12% Bis/Tris polyacrylamide gels in the presence of Mes buffer (A–C).
Arrows indicate the MTB HSP16.3 band. Lane 1 of each gel contains the molecular mass markers. Each individual lane is a sample of MTB
HSP 16.3 plus 0.51 lg chymotrypsin taken at 5-min intervals over a 30-min period. MTB HSP16.3 was readily degraded by chymotrypsin and
nearly all i ntact protein was degraded by 15 min (A). In the presence of 3.5 m
M
ATP, MTB HSP16.3 was stabilized against proteolysis by
chymotrypsin and intact MTB HSP16.3 remained after 30 min of digestion (B). In the presence of the non hydrolyzable analogue of ATP, ATPcS,
there was no stabilization of MTB HSP16.3 against chymotrypsin proteolysis (C), consistent with the effect of ATPcS on the molecular chaperone
function of MTB HSP16.3 reported i n F ig. 3.
Ó FEBS 2002 Similaritiesbetween MTB HSP16.3andaB-crystallin (Eur. J. Biochem. 269) 1811
chaperone function was enhanced by ATP [23]. In separate
reports, ATP increased the refolding of xylose reductase by
total a-crystallin [34], increased the binding of a-crystallin to
lens membranes, and inhibited the chaperone activity of a
plant sHsp [35,36]. C onsistent with previous studies using
aB-crystallin, the present results demonstrated that ATP
enhanced the chaperone effect of MTB HSP16.3 by
twofold in the CS aggregation assay. However, the specific
role of ATP in t he chaperone function of sHsps has been
controversial [18,23,28,29]. Previous reports indicated that
the chaperone activity of MTB H SP 16.3 may be ATP
independent [13,19] while structural studies demonstrated
an interaction between ATP and total bovine a-crystallin
using equilibrium binding studies, intrinsic tryptophan
fluorescence and
31
P NMR [23,25,34,35,37]. aB-Crystallin
has a lso been reported to d isplay an autok inase activity
[38–40]. R ecent r eports suggest that A TP may participate in
the release of tar get peptides from aA-crystallin [24,25].
Here, M TB HSP16.3 chaperone activity was measured in a
Tris/HCl buffer system, while previous studies of MTB
HSP 16.3 were performed in either a Hepes/HCl or sodium
phosphate buffer systems [13,19]. The conditions used in
this study were the same as those used successfully to
demonstrate the ATP effect on humanaB-crystallin [23].
In separate experiments the chymotrypsin proteolytic
digestion pattern of MTB H SP 16.3 in the presence and
absence of ATP was evaluated. Similar to aB-crystallin [28]
and Hsp27 [32], chymotrypsin cleavage sites in MTB
HSP 16.3 appeared to be shielded in the presence of ATP.
The s imilarity of the chymotrypsin d igestion pattern for
MTB HSP16.3 to previous studies with aB-crystallin and
Hsp27 may indicate similar domain structures and assembly
properties that are stabilized in the presence of ATP. As
with aB-crystallin, ATPcS (a nonhydrolyzable A TP analog)
did not enhance the chaperone function of MTB HSP 16.3,
and did not protect against i ts proteolysis by c hymotrypsin.
Although there is on ly 18% sequen ce identity, the c ore
Ôa-crystallinÕ domain in M TB HSP16.3 may have functional
significance similar to that of aB-crystallin [28].
MTB HSP16.3 of M. tuberculosis may be ideally suited
for studies of the structure and function of the core
Ôa-crystallinÕ domain of sHsps because the quaternary
structure is more monodisperse than aB-crystallin and
other sHsps, that are known to have highly variable
quaternary structures [ 41]. T he crystal s tructure of HSP
16.5 from Methanococcus janaschii demonstrates a mono-
mer containing a core domain that consists largely of
b sheets [42]. The molecules o f HSP 16.5 form dimers that
assemble into a spherical complex of octahedral symmetry,
while MTB HSP16.3 is reported t o consist of a trimer of
trimers [13]. Spin labeling o f MTB HSP16.3 in solution is
consistent with a core domain consisting of a twofold
symmetric interface b etween subunits that involved two
b strands in the core a-crystallin domain interacting in an
antiparallel fashion [43]. While previou s mutagenesis studies
demonstrated thefunctional importance of the core
Ôa-crystallinÕ domain in s Hsps [28,29,33], the structural basis
for the function of the conserved core Ôa-crystallinÕ domain
remains to be defined.
New strategies are needed to understand the precise
mechanism that allows the tubercle bacilli of M. tuberculosis
to survive long-term dormancy. This study supports the
hypothesis t hat the conserved core Ôa-crystallinÕ domain i n
MTB HSP16.3 may be important for long-term dormancy
in M. tuberculosis.Futurein vivo studies of specific inter-
actions between MTB HSP16.3and other latent stage
proteins will lead to a b etter u nderstanding of the molecular
chaperone activity of MTB HSP 16.3. Further structure–
function analyses including the determination of an atomic
resolution model of MTB HSP16.3 are needed. Crystallo-
graphic studies of HSP16.3 could be used to determine sites
for interactions with other proteins and/or ATP. Structure–
function studies on M TB HSP16.3 may have important
implications for therapeutic drug discovery for the eradica-
tion of bacilli in the latent s tage of human M. t uberculosis
infection.
ACKNOWLEDGEMENTS
We thank H. Mchaourab for the kind gift of the H SP 16.3 clone,
D. Sherman for the kind gift of the monoclonal antibody IT-4
(a-16 kDa), an d S. Yarfitz for technical assistance with the Mu ltalin
Sequence a lignment program. We also thank J . Clark and C . Ganders
for technical assistance. This work was supported by National Eye
Institute Grant E Y0452 (to J. I. C.).
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Ó FEBS 2002 Similaritiesbetween MTB HSP16.3andaB-crystallin (Eur. J. Biochem. 269) 1813
. HSP 16. 3. [d,CSalone;, ,HSP1 6 .3: CS (10:1)+1m
M
KCl and 3. 5 m
M
MgCl
2
; s, HSP 16. 3 : CS (10 : 1); . ,HSP 16. 3 : CS
(10 : 1) + 3. 5 m
M
ATPcS; j, HSP 16. 3. Functional similarities between the small heat shock proteins
Mycobacterium tuberculosis
HSP 16. 3 and human aB-crystallin
Melissa