Báo cáo khoa học: Conformational properties of bacterial DnaK and yeast mitochondrial Hsp70 Role of the divergent C-terminal a-helical subdomain pdf

Conformational properties of bacterial DnaK and yeast
mitochondrial Hsp70
Role of the divergent C-terminal a-helical subdomain
Fernando Moro
1
, Vanesa Ferna
´
ndez-Sa
´
iz
1
, Olga Slutsky
2
, Abdussalam Azem
2
and Arturo Muga
1
1 Unidad de Biofı
´
sica (CSIC-UPV ⁄ EHU) y Departamento de Bioquı
´
mica y Biologı
´
a Molecular, Universidad del Paı
´
s Vasco, Bilbao, Spain
2 George S. Wise Faculty of Sciences, Department of Biochemistry, Tel Aviv University, Israel
Molecular chaperones of the Hsp70 family are ubiquit-
ous proteins that perform functions essential for cel-
lular life, including protein folding, the assembly of
protein complexes, protein degradation, the transloca-

tion of proteins across membranes and regulation of
the heat shock response [1]. To carry out these differ-
ent functions, Hsp70s rely on the ability to bind short
hydrophobic peptide stretches in extended conforma-
tions that might become accessible within the sequence
of a protein. Conservation among different members
of the family is high and extends to both sequence and
structure, as revealed by the available three-dimen-
sional structures of isolated protein domains [2–6].
Thus, Hsp70s are composed of a highly homologous
N-terminal ATPase domain of  45 kDa, connected
by a short linker to a more variable peptide-binding
domain (PBD) of 25 kDa, consisting of a conserved
b-sandwich and a more variable a-helical subdomain
[7]. The latter subdomain forms a lid that closes the
binding site without contacting the peptide substrate
[4,5]. The peptide binding site consists of a hydro-
phobic cavity formed by loops that protrude from the
Keywords
allosterism, chaperones, DnaK,
mitochondrial Hsp70
Correspondence
F. Moro or A. Muga, Unidad de Biofı
´
sica
(CSIC-UPV ⁄ EHU) y Departamento de
Bioquı
´
mica y Biologı
´

a Molecular, Facultad
de Ciencias, Universidad del Paı
´
s Vasco,
Apartado 644, 48080 Bilbao, Spain
E-mail: or

(Received 8 April 2005, revised 20 April
2005, accepted 25 April 2005)
doi:10.1111/j.1742-4658.2005.04737.x
Among the eukaryotic members of the Hsp70 family, mitochondrial Hsp70
shows the highest degree of sequence identity with bacterial DnaK.
Although they share a functional mechanism and homologous co-chaper-
ones, they are highly specific and cannot be exchanged between Escherichia
coli and yeast mitochondria. To provide a structural basis for this finding,
we characterized both proteins, as well as two DnaK ⁄ mtHsp70 chimeras
constructed by domain swapping, using biochemical and biophysical meth-
ods. Here, we show that DnaK and mtHsp70 display different conforma-
tional and biochemical properties. Replacing different regions of the DnaK
peptide-binding domain with those of mtHsp70 results in chimeric proteins
that: (a) are not able to support growth of an E. coli DnaK deletion strain
at stress temperatures (e.g. 42 °C); (b) show increased accessibility and
decreased thermal stability of the peptide-binding pocket; and (c) have
reduced activation by bacterial, but not mitochondrial co-chaperones, as
compared with DnaK. Importantly, swapping the C-terminal a-helical sub-
domain promotes a conformational change in the chimeras to an mtHsp70-
like conformation. Thus, interaction with bacterial co-chaperones correlates
well with the conformation that natural and chimeric Hsp70s adopt in
solution. Our results support the hypothesis that a specific protein structure
might regulate the interaction of Hsp70s with particular components of the

cellular machinery, such as Tim44, so that they perform specific functions.
Abbreviations
DSC, differential scanning spectroscopy; DTT, dithiothreitol; GdnHCl, guanidine hydrochloride; IR, infrared spectroscopy; mtHsp70,
mitochondrial Hsp70; PBD, peptide binding domain.
3184 FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS
b-sandwich, its accessibility being controlled by the lid
subdomain. Despite the high sequence and structural
homology, Hsp70s are highly specific and the basis of
this functional specificity is not well understood. It
has been postulated that different substrate specificities
and cellular factors such as co-chaperones might be
related to the functional diversification of Hsp70 pro-
teins [8–11].
Among bacterial and eukaryotic Hsp70 proteins,
DnaK of Escherichia coli and mitochondrial Hsp70
(mtHsp70 or Ssc1 in Saccharomyces cerevisiae) are the
two members with the highest degree of sequence con-
servation [12,13], and are thought to share a similar
functional mechanism. Thus, DnaK and mtHsp70
cooperate with the co-chaperones DnaJ and GrpE in
bacteria, and with Mdj1p and Mge1p in yeast mito-
chondria, respectively [14–17]. Despite the homology,
DnaK and mtHsp70 are not interchangeable in bac-
teria or yeast [18,19]. In the mitochondrial matrix,
mtHsp70 is engaged in mitochondrial preprotein trans-
location, a function absent in the bacterial cytosol.
mtHsp70 is recruited to the mitochondrial inner mem-
brane import machinery by Tim44, an essential com-
ponent of the TIM23 complex [20], forming the import
motor that facilitates translocation of precursors across

the inner membrane by a nucleotide-dependent mech-
anism [21]. In vitro, DnaK is able to interact with mito-
chondrial presequences [22], indicating that substrate
affinities of DnaK and mtHsp70 are similar. When
expressed in the mitochondrial matrix, DnaK is able
to interact with Tim44, their interaction not being
regulated by nucleotides, and the complex is not able
to promote the import of precursors [18].
To gain further insight into conformational differ-
ences between DnaK and mtHsp70 that might be
important for the functional specificity within this
protein family, we purified both proteins from E. coli
and yeast mitochondria, and characterized their bio-
chemical and biophysical properties. In addition, we
studied the chimeras KKCC and KCCC constructed
by domain swapping [18] (Fig. 1A). While maintain-
ing the ATPase domain of DnaK, different regions of
the more divergent substrate-binding domain were
exchanged: (a) a-helical subdomain and C-terminal
residues in KKCC; and (b) complete PBD in KCCC.
Our results indicate that in spite of the expected
structural similarity, these proteins show different
conformational properties that affect their interaction
with peptide substrates, bacterial co-chaperones, and
their ability to refold denatured substrates, suggesting
that the particular conformation that members of the
Hsp70 family might adopt could be related to their
functional specificity.
A
B

Fig. 1. (A) Outline of DnaK ⁄ mtHsp70 chimeras. DnaK and mtHsp70
sequences are represented by white and black boxes, respectively.
Fusion points are indicated according to the numbering of DnaK
residues. Identity values obtained with
CLUSTALW are given in bold.
The source of the corresponding domain or subdomain is specified
by K (DnaK) and C (mtHsp70), and the chimeric proteins are named
following a previously reported nomenclature [18]. (B) Peptide and
co-chaperone-induced ATPase activity stimulation. S teady-state
ATPase was measured at 30 °C, protein and ATP concentrations
were 5 l
M and 1 mM, respectively. NRLLLTG (NR) peptide was
added at 0.5 m
M. DnaJ and GrpE concentrations were 0.5 lM and
1.5 l
M, respectively. Mdj1p and Mge1p were used in the same
concentration as DnaJ and GrpE. Specific activity values (upper)
and ratio of the Hsp70 ATPase activity in the presence of the speci-
fied ligands to the activity without co-chaperones or peptide sub-
strate (lower).
F. Moro et al. Hsp70 structure and specificity
FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS 3185
Results
Chimeric Hsp70s are not able to complement
DnaK function in vivo
To study the functionality of chimeric Hsp70s, we per-
formed complementation experiments in the E. coli
temperature-sensitive DdnaK52 strain BB1553 [23].
None of the chimeras studied here was able to support
growth at 42 °C (not shown). It should be mentioned

that yeast mtHsp70, also termed Ssc1p, does not
support the growth of an E. coli DnaK-deletion strain
[19]. Furthermore, coexpression of mtHsp70 and
Mdj1p did not suppress the temperature-sensitive bac-
terial phenotype, indicating that the Hsp70 representa-
tive is not interchangeable, because Mdj1p can replace
DnaJ [19]. Moreover, none of the chimeras was able to
complement the deletion of the ssc1 gene in the yeast
S. cerevisiae [18].
Allosteric stimulation of ATPase activity by
peptide substrates and co-chaperones
Hsp70 proteins are ATPases that are allosterically sti-
mulated by substrate binding. Therefore, this stimu-
lation can be used as a signature of interdomain
communication (Fig. 1B). Peptide NRLLLTG (NR)
was chosen as the substrate because it binds both
DnaK and mtHsp70 with high affinity [24,25]. Consis-
tent with previous observations [17,26], wild-type
mtHsp70 showed a steady-state ATPase activity signifi-
cantly higher than DnaK (0.3 and 0.1 mol ATPÆ
mol protein
)1
Æmin
)1
, respectively). Upon addition of
NR peptide, mtHsp70 was stimulated 2.5 times,
whereas DnaK underwent a fivefold stimulation, in
good agreement with previous studies [26,27]. Lower
activation of mtHsp70 (fivefold) was also achieved by
bacterial co-chaperones (DnaJ and GrpE), compared

with DnaK (20-fold). Defective activation by E. coli
DnaJ and GrpE has also been reported for Vibrio har-
veyi DnaK [28]. In contrast, mitochondrial co-chaper-
ones (Mge1p and Mdj1p) similarly stimulate the
ATPase activity of both Hsp70 proteins (five- to six-
fold). It should be mentioned that a 20-fold activation
of mtHsp70 by Mdj1p and Mge1p can be achieved at
different molar ratios [26] than those used in this study
(10 : 1 : 3, see Experimental procedures). These were
chosen because they seem to be closer to the physiolo-
gical molar ratio [29,30]. The steady-state ATPase
activities of KKCC and KCCC were comparable with
that of DnaK, however, addition of the NR peptide
poorly enhanced their activity (less than twofold). Bac-
terial co-chaperones activate both chimeric proteins
more than their mitochondrial counterparts, an effect
better seen for KKCC. Interestingly, the relative
DnaJ ⁄ GrpE-induced activation observed for KKCC
and KCCC was similar to that found for mtHsp70.
Taken together, the data indicate that chimeric pro-
teins behave as mtHsp70 regarding the stimulation
of their ATPase activities by peptide substrates and
co-chaperones. As expected, this behavior becomes
more similar when the whole peptide domain of DnaK
is replaced by mtHsp70 sequence.
Substrate-binding properties and refolding
activity
We next investigated the ability of natural and chi-
meric Hsp70s to bind peptide substrates. Binding
of fluorescein-CALLQSRLLLSAPRRAAATARY (F-

APPY) was monitored through changes in fluorescence
anisotropy as described elsewhere [27]. Equilibrium
binding curves were performed with increasing concen-
trations of Hsp70 proteins, and the anisotropy increase
was fitted to a single site binding model (Fig. 2A;
Table 1). The affinity of these proteins for F-APPY
was similar (Table 1), as also indicated by kinetic
measurements (see below). The allosteric communica-
tion between the ATPase domain and PBD of chimeric
Hsp70s was functional, because preformed F-APPY
complexes were rapidly dissociated upon addition of
ATP (not shown).
We also characterized the binding kinetic parameters
k
+1
and k
)1
. F-APPY binding kinetics were followed
at increasing protein concentrations and were fitted to
a single exponential compatible with a single site bind-
ing model (not shown). The plots of k
obs
against pro-
tein concentration were linear and k
+1
and k
)1
were
derived from the y-intercept and the slope, respectively
(Table 1) [31]. Comparison of wild-type DnaK and

mtHsp70 showed that the binding constants of the lat-
ter are around threefold higher, suggesting a higher
accessibility of its binding pocket. A significantly
higher increase is observed for KKCC (twelve- and
eightfold for k
+1
and k
)1
, respectively) and KCCC
(eight- and sixfold), suggesting that the lid did not
close tightly the binding site of these chimeras. Thus,
this finding indicates that the interaction of mtHsp70
PBD with the DnaK ATPase domain modifies the
accessibility of the substrate binding site.
The thermal stability of the peptide complexes of
DnaK, mtHsp70 and chimeric proteins was also char-
acterized by fluorescence spectroscopy. F-APPY bind-
ing kinetics were analyzed at 25, 37 and 42 °C in the
presence of excess protein (Fig. 2B). As observed pre-
viously for DnaK [32], mtHsp70 binding kinetics were
Hsp70 structure and specificity F. Moro et al.
3186 FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS
faster at higher temperatures. It should be noted that
the temperature-induced destabilization of the peptide-
bound complex was more pronounced for mtHsp70.
KKCC (not shown) and KCCC, however, bound
F-APPY much faster than the wild-type proteins at
25 °C, as observed for lidless mutants [31,32] (Fig. 2B),
and showed a significantly reduced binding at 37 °C
that is virtually abolished at 42 °C (Fig. 2C). As also

found for lidless mutants of DnaK [32], cooling the
samples completely restored binding (not shown),
suggesting that the temperature-induced conformat-
ional change responsible for the reduced binding was
reversible.
Finally, we studied the ability of natural and chi-
meric Hsp70s to refold chemically and thermally
denatured luciferase (Fig. 3). Only DnaK was able to
refold luciferase denatured by guanidine hydrochlo-
ride (GdnHCl), the reactivation yield being highly
sensitive to the source of the co-chaperones (Fig. 3A).
The percentage of reactivated luciferase decreased
from 60 to 25% when bacterial co-chaperones were
replaced by their mitochondrial homologs. Because
mtHsp70 requires Hsp78, the mitochondrial ClpB
(Hsp100) homolog, to refold chemically denatured
luciferase [33], we next tried to follow Hsp70-medi-
ated reactivation of thermally denatured luciferase, a
process that mtHsp70 can perform with only the help
of its co-chaperones [30]. Both natural Hsp70s effi-
ciently refold the substrate protein that was progres-
sively denatured in the presence of the chaperones
(Fig. 3B). However, the refolding yield of DnaK, in
contrast to mtHsp70, decreases significantly (from
 80 to 37%) when using mitochondrial instead of
bacterial co-chaperones, as also observed with chemic-
ally denatured luciferase. Compared with natural
Hsp70s, the refolding efficiency of the chimeras was
half in the presence of bacterial co-chaperones, and
replacement of these proteins by their mitochondrial

homologs did not significantly modify the reactivation
percentage. This reduction might be due to the lower
stability of the peptide–chimera complexes and ⁄ or
to their lower ATPase activity. Nevertheless, these
results also suggest that chimeras might chaperone
protein folding in vitro.
A
BC
Fig. 2. Peptide interaction properties of DnaK, mtHsp70 and chime-
ras. (A) F-APPY binding curves were performed at a fixed peptide
concentration of 35 n
M and varying Hsp70 concentrations: DnaK,
d; mtHsp70, h; KKCC, ,; KCCC, n. Samples were incubated
overnight at 4 °C to achieve equilibrium and left at 25 °Cfor2h
before measuring the anisotropy value. Solid lines represent the
best fit of data to a single site binding model. (B) F-APPY binding
kinetics of DnaK (upper), mtHsp70 (middle) and KCCC (lower) at
25, 37 and 42 °C. Binding was carried out in the presence of
0.5 m
M ADP to avoid thermal denaturation of DnaK ATPase domain
at 42 °C [42]. The reaction was initiated by addition of F-APPY
(35 n
M final concentration) to a thermostated solution of protein
(1 l
M). (C) Anisotropy increment at the saturation plateau for DnaK,
mtHsp70 and KCCC at 25, 37 and 42 °C. Values were obtained
after fitting the experimental data to single exponential curves.
Table 1. F-APPY dissociation and binding constants. k
+1
and k

)1
were obtained at 25 °C.
K
d
(lM) k
+1
(M
)1
Æs
)1
) k
)1
(s
)1
· 10
3
)
DnaK 0.115 (± 0.007) 570 0.60
KKCC 0.271 (± 0.011) 6980 5.06
KCCC 0.165 (± 0.009) 4290 3.32
mtHsp70 0.196 (± 0.025) 1940 1.93
F. Moro et al. Hsp70 structure and specificity
FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS 3187
In summary, the affinity of the wild-type proteins
and the chimeras for F-APPY was reasonably similar
at 25 °C. Sequence substitutions in KKCC and KCCC
affected the binding kinetics, the thermal stability of
Hsp70–peptide complexes, and the refolding activity
which might reflect a destabilization of the PBD.
Conformational properties of wild-type and

chimeric proteins: fluorescence and infrared
spectroscopy
In order to rule out possible misfolding of the chimeric
proteins, their secondary structure was characterized
by infrared spectroscopy (IR). As shown previously
[32,34], the amide I band of DnaK showed an absorp-
tion maximum at 1650 cm
)1
in aqueous buffer
(Fig. 4A). After deconvolution, several band compo-
nents representing the different types of secondary
structures in the protein were observed, whose assign-
ment has been described [32]. The IR spectra of DnaK,
mtHsp70 and chimeric proteins were similar regarding
both the number and position of their amide I compo-
nents (Fig. 4A; for the sake of simplicity, only spectra
of KCCC are shown). Furthermore, decomposition of
the amide I band into its components indicated that
the relative area of each component was similar, within
experimental error, for all proteins. This finding is in
good agreement with circular dichroism studies show-
ing a similar secondary structure for DnaK and
mtHsp70 [35], and also indicates that sequence
exchange at the PBD of DnaK did not modify the
overall secondary structure of the chimeras.
The intrinsic fluorescence of DnaK has been widely
used to follow allosteric conformational changes upon
nucleotide binding [27,34,36,37]. Binding of ATP to
DnaK promoted quenching of the single Trp residue
of DnaK and a blue-shift of its emission maximum

(Fig. 4B, upper traces). In addition to these spectral
changes, reduction of the tryptophan accessibility to
polar quenchers was observed upon ATP binding [36]
(Table 2). As previously discussed, these spectroscopic
changes require the interaction of both protein
domains to occur, and are therefore indicative of allo-
steric communication. A similar quenching effect was
observed for mtHsp70 although the shift of the emis-
sion maximum was not as clearly observed (Fig. 4B,
middle spectra). It should be noted that in the absence
of nucleotide the emission maximum of mtHsp70 is
downshifted by 5–6 nm with respect to that of DnaK.
The twofold reduction of the K
SV
values estimated for
mtHsp70 both in the absence and the presence of ATP
(Table 2) supports the existence of differences in the
Trp environment of this protein and DnaK. Results
obtained for the chimeras (Fig. 4B lower traces, only
emission spectra of KCCC are shown; Table 2), indi-
cate that they undergo nucleotide-induced conforma-
tional changes similar to those observed for DnaK.
Partial proteolysis and stability: sequence
exchange modifies the tryptic sites topology and
protein stability
Partial proteolysis gives a valuable indication of pro-
tein tertiary structure because the accessibility of tryp-
tic sites depends on protein conformation. DnaK has a
very characteristic pattern of tryptic fragments [37,38]
(Fig. 5A), most of the tryptic sites at the C-terminal

domain being sensitive to ATP as a consequence of
interdomain allosteric coupling. In the absence of nuc-
leotide or in the presence of ADP, tryptic fragments
with apparent molecular masses of 55, 46, 44, 33 and
31 kDa were generated, the 44- and 31-kDa fragments
being predominant at 15 min and longer times of pro-
teolysis. In the presence of ATP, DnaK was degraded
faster and the fragment pattern changed significantly:
(a) a new 53 kDa fragment was generated at short
A
B
Fig. 3. Refolding activity of natural and chimeric Hsp70s in the
presence of bacterial or mitochondrial co-chaperones. Reactivation
of GdnHCl-denatured (A) or heat-treated (B) luciferase by the indica-
ted Hsp70 protein in the presence of DnaJ ⁄ GrpE (black bars) or
Mdj1p ⁄ Mge1p (gray bars). See Experimental procedures for protein
concentrations. Control refers to refolding in the absence of chaper-
ones (white bars).
Hsp70 structure and specificity F. Moro et al.
3188 FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS
times; (b) the 33 and 31 kDa species were found only
in small proportions; (c) the 46-kDa band was pre-
dominant at short and intermediate times, and (d)
after 40 min, the 44 kDa band was the most abundant.
In the absence of nucleotides or in the presence of
ADP, KKCC and KCCC generated mainly 46 and
44 kDa fragments in an approximately 1 : 1 ratio
(Fig. 5B,C, respectively), their formation being
strongly reduced in the presence of ATP. The ATP-
bound state of both chimeras was more resistant to

trypsin, as reported for Hsp70 and Bip [39,40], and
predominantly gave rise to a fragment of 58 kDa.
Assignment of the site that gives rise to the 58 kDa
fragment is difficult due to the low conservation of the
replaced sequence, however, the 44 and 46 kDa frag-
ments have been related to two sites located in the lin-
ker connecting the ATPase and PBD of DnaK [37].
Both sites are conserved in the KKCC and KCCC
sequences (Fig. 5E), indicating that the accessibility of
the linker region to trypsin in both chimeras was
reduced in the presence of ATP, compared with DnaK.
mtHsp70 also gave rise to a 58 kDa fragment in the
presence of ATP (Fig. 5D), as shown previously in
total lysates of mitochondria [41]. However, the
44 kDa band, possibly corresponding to the ATPase
domain, was generated regardless of the bound nucleo-
tide. The absence of a proteolytic 46-kDa fragment
with mtHsp70 might be due to the loss of this tryptic
site (Fig. 5E). That these chimeras give rise to similar
proteolytic patterns indicates that, in spite of sequence
differences, they both adopt a similar conformation
that is, in turn, distinct from DnaK. Furthermore, the
change in accessibility of tryptic sites suggests that
replacement of the C-terminal sequences in KKCC and
KCCC promotes an alteration of the protein tertiary
structure that becomes similar to that of mtHsp70.
Table 2. Apparent Stern–Volmer constants (K
sv
, M
)1

) obtained in
the absence and presence of 0.5 m
M ATP. K
sv
were determined
from the equation F
o
⁄ F ¼ 1+K
sv
· [acrylamide]. Data are the aver-
age of at least three independent experiments on two different pro-
tein batches.
Free +ATP
DnaK 9.64 3.24
KKCC 9.62 3.54
KCCC 10.57 3.94
mtHsp70 4.7 2.3
Fig. 4. Spectroscopic properties of wild-type DnaK, mtHsp70 and chimeric Hsp70. A. IR spectra of DnaK, mtHsp70 and KCCC. Spectra were
recorded in 100 m
M Mops, pH 7.0, 50 mM KCl, 10 mM MgCl
2
. Protein concentration was 30–40 mgÆmL
)1
. Thick and thin solid lines repre-
sent the original and deconvoluted spectra for each protein, respectively. Deconvolution was performed using a Lorentzian band-width of
18 cm
)1
and a resolution enhancement factor of two. (B) Fluorescence emission spectra of DnaK (upper), mtHsp70 (middle) and KCCC (bot-
tom) recorded in the absence of nucleotides (solid line) and in the presence of 0.5 m
M ATP (broken line). Protein concentration was 5 lM.

F. Moro et al. Hsp70 structure and specificity
FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS 3189
Next, the thermal stability of DnaK, mtHsp70 and
the chimeras was studied by differential scanning
spectroscopy (DSC; Fig. 6, Table 3). As previously
reported [42], thermal unfolding of DnaK gave rise to
three endotherms at 43.5, 57.5 and 74 °C. The first
and second endotherms have been assigned to the
unfolding of the N-terminal ATPase domain and the
C-terminal PBD, respectively, whereas the third con-
tains contributions from the denaturation of both
domains [42]. It should be noted here that the reversi-
bility of the thermal denaturation process of all
Hsp70s was the same as that described for wild-type
DnaK [42]. Replacement of the complete a-helical
domain in KKCC resulted in disappearance of the
intermediate endotherm, which was assigned to the
PBD. In contrast to what was observed for the other
proteins, the experimental DSC profile of this chimera
was better fitted with four transitions (Fig. 6,
Table 3). Whereas three of these transitions appear at
temperatures similar to those found for the ATPase
domain of DnaK, and what might be, by analogy
with DnaK, the PBD of mtHsp70, assignment of the
small (12.5 kcalÆmol
)1
) endotherm at 51.8 °C is not
straightforward. It may represent the denaturation of
a destabilized folding unit either at the PBD or at an
interdomain region. The second alternative would be

supported by the fact that it completely disappears in
the DSC trace of KCCC, or if a residual one
remained, it completely merged with the peak corres-
ponding to the unfolding of the ATPase domain. The
overall destabilization associated with sequence
exchange is shown by the enthalpy values of the over-
all denaturation process: 245, 135.5 and 159 kcalÆ
mol
)1
for DnaK, KKCC and KCCC, respectively
(Table 3). Thermal denaturation of mtHsp70 also
showed three endotherms centered at 51.6, 67.5 and
76.2 °C (Table 3) with an overall denaturation
enthalpy of 214 kcalÆmol
)1
. Although a detailed study
would be needed to assign the experimental endo-
therms to the unfolding of the corresponding
mtHsp70 domains, it is reasonable to propose that
the endotherm at 51.6 °C could represent the unfold-
ing of a more stable ATPase domain. The T
m
values
of the high-temperature endotherms, which are similar
to those of KKCC and KCCC and clearly distinct
from those of DnaK, suggest that the stabilizing
A
B
C
D

E
Fig. 5. KKCC and KCCC tryptic sites have an altered topology. Coo-
massie Brilliant Blue-stained SDS ⁄ PAGE of tryptic fragments of (A)
DnaK, (B) KKCC, (C) KCCC, (D) mtHsp70. Partial tryptic proteolysis
was carried out at 30 °C in the absence or presence of nucleotide
(1 m
M final concentration). Aliquots were taken at different times
and analyzed. Three micrograms of protein and 0.15 lg trypsin
were loaded on each lane. (E) Sequence alignment of putative tryp-
tic sites of the proteins studied. Sites were taken from Fig. 6 of
Buchberger et al. [37].
Hsp70 structure and specificity F. Moro et al.
3190 FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS
interactions within the PBD domain of these proteins
are different, and that substitutions in KKCC and
KCCC promote a domain organization that resembles
that of mtHsp70.
Discussion
DnaK and mtHsp70 share a high degree of primary
sequence conservation, as expected from the prokary-
otic origin of mitochondria [12,13]. They are thought
to have a similar mechanism for binding unfolded
polypeptides and cooperate with homologous co-chap-
erones of the Hsp40 family (DnaJ and Mdj1p, respect-
ively) and a nucleotide exchange factor (GrpE and
Mge1p). Despite these similarities, DnaK and
mtHsp70 are specific and cannot be exchanged
between E. coli and S. cerevisiae mitochondria [18,19].
Although cross-species complementation cannot be
attributed to a single factor, it should be mentioned here

that mitochondria have developed a Hsp70-dependent
import motor for nuclear-encoded mitochondrial pro-
tein, a function absent in bacteria. Similarly, an
increasing number of biochemical and genetic studies
have addressed the functional nonequivalence of
Hsp70 chaperones from different sources [43–45]. Two
arguments have been put forward to explain the diver-
sification and functional specificity of the Hsp70 chap-
erone system: (a) a different ability to interact with
specific co-chaperones [11,46], and (b) changes in sub-
strate affinity due to modifications of the substrate-
binding site and ⁄ or changes in the dynamics of the lid
[9,45]. In fact, both substrate affinity and interaction
with specific co-chaperones might be related to the
conformational properties of an Hsp70 protein. In this
context, our data provide new experimental evidence
of a distinct conformation of DnaK and mtHsp70, in
spite of their similar overall secondary structure. The
results presented here are discussed taking into account
the above arguments and the conformational differ-
ences observed between DnaK and mtHsp70.
A different interaction with co-chaperones is inferred
according to the observed three- to fourfold lower bac-
terial co-chaperone-induced stimulation of the ATPase
activity of mtHsp70 and the chimeric proteins, com-
pared with DnaK. Considering that GrpE and DnaJ
most likely interact with sites located at both the
N-terminal ATPase domain and the PBD of DnaK
[3,47–49], the putative binding site(s) at the PBD of
mtHsp70 and the chimeras could be modified as a con-

sequence of sequence exchange and ⁄ or a distinct con-
formation due to a sequence-specific folding of this
domain. Proteolysis and DSC results clearly show that
the chimeras KKCC and KCCC fold into a similar
tertiary structure, but different from that of DnaK,
whereas their secondary structure, as seen by IR
spectroscopy, remains similar. However, the fact that
this difference is not observed, under the same experi-
mental conditions, with mitochondrial co-chaperones
Fig. 6. Thermal stability of wild-type and chimeric Hsp70s. Calori-
metric traces of the different proteins in 25 m
M Glycine, pH 9.0 at
1–2 mgÆmL
)1
protein concentration. The scan rate was 60 °CÆh
)1
.
Open circles represent the experimental points, dashed lines repre-
sent the result of the best fit obtained from deconvolution analysis
assuming a three-transition model, and thick solid lines represent
the overall fit.
F. Moro et al. Hsp70 structure and specificity
FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS 3191
indicates that they interact differently with DnaK, as
also seen in refolding assays (Fig. 3).
Peptide-binding properties show that sequence sub-
stitutions result in chimeric proteins with an increased
accessibility and decreased thermal stability of the pep-
tide-binding site. Similar findings have been reported
for DnaK deletion mutants lacking helices A and B of

the lid subdomain [31,32], suggesting that the stabili-
zing interactions between residues at aB and the loops
forming the binding site are not properly established in
these chimeras. Comparison of the binding constants
of chimeric and wild-type proteins also indicates that
the stability of the peptide-binding pocket of mtHsp70
depends on the presence of its ATPase domain,
because KCCC does not interact with peptide sub-
strates at stress temperatures, e.g. 42 °C. Therefore,
the functionality of the peptide-binding site depends
on interactions between the b-sandwich and both the
lid subdomain and the ATPase domain of the protein.
This structural organization might reflect an intermedi-
ate role for the b-subdomain in transmitting the allo-
steric signal, which, in the presence of ATP, goes from
the ATPase domain to the helical lid and results in
peptide release. The instability of the substrate-binding
site of both chimeras might also be related to their fail-
ure to significantly refold thermally denatured luci-
ferase. At stress temperatures (e.g. 42 °C) chimeric
proteins could not stably bind denatured luciferase,
which would aggregate in solution. In contrast, native
Hsp70s would interact with denatured luciferase,
avoiding aggregation, and could refold the substrate
once stress conditions disappear. Therefore, the refold-
ing activity of these proteins might also help to explain
why they cannot support growth of a DnaK deletion
strain and yeast lacking or harboring a mutant
mtHsp70 [18].
This brings us to the allosteric behavior of these pro-

teins, because it is well known that proper functioning
of Hsp70 proteins requires interdomain communica-
tion. As judged by the peptide-induced activation of
the ATPase activity, ATP-induced peptide dissociation,
and intrinsic fluorescence data of the chimeras,
sequence exchange does not hamper interdomain com-
munication. However, the response to different ligands
is not identical for wild-type DnaK and chimeric pro-
teins. Note that substrates stimulate the activity of the
chimeras 2–3 times less than that of DnaK, resembling
the activation observed for mtHsp70. As far as sub-
strate-induced ATPase activation is concerned, only
the interaction between the ATPase domain and
b-sandwich is important [31,50]. This suggests that the
interface between the DnaK ATPase domain and
the b-sandwich, whether belonging to DnaK or to
mtHsp70, is modified as a consequence of the substitu-
tion of the divergent a-helical subdomain. Interest-
ingly, the crystal structure of the C-terminal a-helical
subdomain of two Hsp70 proteins, E. coli HscA [5]
and rat Hsc70 [51], indicates that they contain either a
different number of a-helices and ⁄ or distinct interheli-
cal interactions. Thus, the sequence and conformation-
al variability of the a-helical subdomain might be
an important factor for maintaining the conformation
of the whole PBD and modulating the interdomain
interface.
These findings, together with comparison of the
thermal stability, trypsin accessibility and stimulation
by co-chaperones, suggest that exchange of the a-heli-

cal subdomain or the whole PBD promotes a conform-
ational transition of the protein to a mtHsp70-like
conformation. This interpretation would be in agree-
ment with the ability of the chimeras KKCC and
KCCC to interact with the mitochondrial inner mem-
brane protein Tim44 in a nucleotide-dependent man-
ner, as does wild-type mtHsp70 [18]. Although we
cannot rule out a sequence-specific effect on the inter-
action of these proteins with Tim44, specific co-chaper-
ones and protein substrates, we find that this
interaction might be modulated by a conformational
change affecting mainly the exchanged sequence, in
our case the PBD. The results presented here support
the hypothesis that a specific tertiary structure might
regulate the interaction of Hsp70s with certain protein
components of the cellular machinery, and therefore
direct their activities to specific functions.
Table 3. Thermodynamic parameters of DnaK, mtHsp70 and chimeras. T
m
values are reported in °C; DH values are in kcalÆmol
)1
. The uncer-
tainty in the experimental values is ± 0.2 °CforT
m
and 15% for DH.
Transition 1 Transition 2 Transition 3 Transition 4
T
m1
DH
1

T
m2
DH
2
T
m3
DH
3
T
m4
DH
4
DnaK 43.5 79 57.5 94 74.0 72 – –
KKCC 46.6 72 51.4 12.5 64.8 19 75.3 32
KCCC 46.0 92 – – 65.3 37 76.3 30
mtHsp70 51.6 120 – – 67.5 70 76.2 24
Hsp70 structure and specificity F. Moro et al.
3192 FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS
Experimental procedures
Cloning and protein purification
KKCC and KCCC chimeras were amplified by PCR from
their corresponding yeast expression vectors [18], using
the primers: 5¢-CCCGCCATGGGTAAAATAATTGGTA
TCG-3¢ and 5¢-CCCGGATCCAAGCTTTTACTGCTTAG
TTTCACCAGA-3¢. The PCR fragments were cloned into
the bacterial expression vector pTrc99A (Amersham Phar-
macia Biotech, Piscataway, NJ) following the protocol des-
cribed for DnaK [36]. Chimeric Hsp70s were overexpressed
in BB1553 cells [23], grown at 30 °C, after isopropyl thio-b-
d-galactoside induction in the exponential phase. After cell

lysis, protein purification was achieved by ion exchange,
ATP-agarose affinity and hydroxyapatite chromatographies
as described previously [36]. MtHsp70 was overexpressed in
the yeast strain YKN3B and purified as described previ-
ously [35]. All proteins were extensively dialyzed against
20 mm imidazole, pH 7.2, 2 mm EDTA, 10% glycerol to
remove the bound nucleotide.
DnaJ and GrpE were expressed in BL21 cells and puri-
fied as described elsewhere [52,53]. Recombinant his-tagged
versions of Mdj1p and Mge1p, where the mitochondrial
presequences were removed, were expressed in E. coli and
purified as described elsewhere [17].
ATPase activity
Steady-state ATPase activity measurements were performed
in 40 mm Hepes, pH 7.5, 50 mm KCl, 11 mm Mg acetate
buffer at 30 °C, as described previously [36]. Protein and
ATP concentrations were 5 lm and 1 mm, respectively.
Reactions were followed measuring the absorbance decay
at 340 nm for 30 min in a Cary spectrophotometer (Var-
ian). In the peptide stimulation assays, NRLLLTG (NR)
peptide was added at 500 lm. GrpE and Mge1p were added
at 1.5 lm. DnaJ and Mdj1p were added at 1.5 and 0.5 lm,
respectively.
Peptide binding
Peptide-binding assays were performed in 25 mm Hepes,
pH 7.6, 50 mm KCl, 5 mm MgCl
2
,1mm dithiothreitol
(DTT). The concentration of F-APPY peptide (fluorescein-
CALLQSRLLLSAPRRAAATARY) was 35 nm and that

of Hsp70 varied from 1 nm to 50 lm. Because binding at
submicromolar concentrations was slow, the mixtures were
prepared and left to equilibrate overnight at 4 °C. Fluor-
escence anisotropy measurements were performed on a
SLM8100 spectrofluorimeter (Aminco) with excitation at
492 nm, emission at 516 nm and 8 nm excitation and
emission slit widths. The fraction of peptide bound to the
Hsp70 protein at each point was calculated and the data
were fitted as described previously [27]. Association kinet-
ics at 25, 37 and 42 °C were performed at F-APPY and
Hsp70 concentrations of 35 nm and 1 lm, respectively,
in the buffer described above, including 0.5 mm ADP.
Data were fitted to a monoexponential equation consistent
with a bimolecular reaction Hsp70 + F-APPY , Hsp70Æ
F-APPY and the k
obs
value was plotted against Hsp70
concentration to obtain the binding parameters k
+1
and
k
-1
.
Refolding of chemically and thermally denatured
luciferase
Chemical denaturation
Firefly luciferase (2.5 lm) was denatured for 45 min at room
temperature in 6 m GdnHCl, 100 mm Tris, pH 7.7, 10 mm
DTT. For refolding, luciferase was diluted to 25 n m in
50 mm Tris, pH 7.7, 55 mm KCl, 15 mm MgCl

2
, 5.5 mm
DTT, 0.5 mgÆmL
)1
bovine serum albumin containing an
ATP-regenerating system (4 mm phosphoenolpyruvate and
20 ngÆmL
)1
pyruvate kinase) and chaperones in the following
concentrations: 1 lm Hsp70 (DnaK, mtHsp70, KKCC and
KCCC), 1 lm DnaJ or Mdj1p, and 1.2 lm GrpE or Mge1p.
Reactivation was initiated by addition of 4 mm ATP and
left for 2 h at room temperature. Luciferase activity was
determined in a Sinergy HT (Biotek) luminometer using the
Luciferase Assay System (Promega E1500).
Thermal denaturation
Refolding of thermally denatured luciferase was performed
as described elsewhere [30]. Briefly, 80 nm luciferase was
incubated for 5 min at 25 °C with 2 lm Hsp70 (DnaK,
mtHsp70, KKCC and KCCC) which was preincubated for
15 min at 25 ° C with 4 mm ATP in 25 mm Hepes, pH 7.5,
50 mm KCl, 5 mm MgCl
2
,5mm 2-mercaptoethanol, and
co-chaperones (0.1 lm Mdj1p and 0.25 lm Mge1p, or
0.1 lm DnaJ and 2 lm GrpE). Denaturation was achieved
incubating the mixture for 10 min at 42 °C. Luciferase
activity was measured as above after a 90 min reactivation
period at 25 °C.
Infrared spectroscopy

Proteins were extensively dialyzed against 100 mm Mops,
pH 7.0, 50 mm KCl, 10 mm MgCl
2
and concentrated on
Microcon-30 (Amicon) filters to final concentration of
30–40 mgÆmL
)1
. The filtrates obtained in the last concentra-
tion step were used as references. Samples were placed in a
thermostatted cell, between two calcium fluoride windows
separated by 6 lm spacers. Infrared spectra were recorded
in a Nicolet Nexus 800 spectrometer equipped with a MCT
detector. Data acquisition and analysis were performed as
described previously [32].
F. Moro et al. Hsp70 structure and specificity
FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS 3193
Intrinsic fluorescence measurements
Intrinsic fluorescence spectra were recorded on a SLM8100
spectrofluorimeter (Aminco) with excitation at 295 nm and
emission at 300–400 nm. Excitation and emission slits were
set at 4 nm. The protein and nucleotide concentrations were
5 lm and 0.5 mm, respectively, and the buffer was 25 mm
Hepes, pH 7.6, 50 mm KCl, 5 mm MgCl
2
,2mm DTT. In
the fluorescence-quenching experiments, the emission was
measured at 340 nm upon excitation at 295 nm. Acrylamide
was gradually added to 3 lm protein in the absence or pres-
ence of 0.5 mm nucleotide. Dilution effects were corrected
and the Stern-Volmer constants estimated as described pre-

viously [36,54].
Partial proteolysis by trypsin
Partial tryptic digestion of DnaK, KKCC, KCCC and
mtHsp70 was performed at 30 °Cin40mm Hepes, pH 7.6,
50 mm KCl, 10 mm MgCl
2
, 0.3 mm EDTA, 2 mm DTT.
Hsp70 proteins (4.3 lm) were incubated with 1 mm ATP,
ADP, or without nucleotide for 30 min at 30 °C. Proteoly-
sis was initiated by addition of trypsin to Hsp70 protein at
a ratio 0.05 : 1 (w ⁄ w). Aliquots were taken at the indicated
time interval and the reaction was stopped by addition of
1mm phenylmethylsufonyl fluoride. Degradation products
were analyzed by SDS ⁄ PAGE (12.5% gels) followed by
staining with Coomassie Brilliant Blue.
Differential scanning calorimetry
DSC was performed in a VP-DSC microcalorimeter (Mic-
rocal, Northampton, MA). Prior to DSC experiments, sam-
ples were dialyzed against 25 mm glycine, pH 9.0. Samples
and reference solutions were properly centrifuged to remove
protein aggregates, degassed and loaded into the calori-
meter. Experiments were carried out under positive pressure
to avoid degassing during heating. The calorimetric data
were analyzed using origin software provided with the
calorimeter. Protein concentration was 1–2 mgÆmL
)1
and
the scanning rate was 60 °CÆh
)1
. Experiments were carried

out twice with at least two different protein batches.
Acknowledgements
We thank Celeste Weiss and Adina Niv for advice
with mtHsp70 purification, and Professor Walter
Neupert for the DnaK ⁄ mtHsp70 chimeras. We also
thank Professor F. M. Gon
˜
i, Dr Stefka Taneva and
Dr Gorka Basan
˜
ez for critically reading the manu-
script. This work was supported by the University of
Basque Country (UPV 13505 ⁄ 2001) and Ministerio
de Educacio
´
n y Ciencia (CICYT BMC 2001 ⁄ 0950
and BFU2004-03452). FM and VF were supported
by postdoctoral and predoctoral fellowships, respect-
ively, from the Basque Government. AA was suppor-
ted by the German–Israeli Foundation (grant no.
753).
References
1 Hartl FU (1996) Molecular chaperones in cellular pro-
tein folding. Nature 381, 571–579.
2 Flaherty KM, DeLuca-Flaherty C & McKay DB (1990)
Three-dimensional structure of the ATPase fragment of
a 70K heat-shock cognate protein. Nature 346, 623–628.
3 Harrison CJ, Hayer-Hartl M, Di Liberto M, Hartl F &
Kuriyan J (1997) Crystal structure of the nucleotide
exchange factor GrpE bound to the ATPase domain of

the molecular chaperone DnaK. Science 276, 431–435.
4 Zhu X, Zhao X, Burkholder WF, Gragerov A,
Ogata CM, Gottesman ME & Hendrickson WA (1996)
Structural analysis of substrate binding by the molecular
chaperone DnaK. Science 272, 1606–1614.
5 Cupp-Vickery JR, Peterson JC, Ta DT & Vickery LE
(2004) Crystal structure of the molecular chaperone
HscA substrate binding domain complexed with the
IscU recognition peptide ELPPVKIHC. J Mol Biol 342,
1265–1278.
6 Wang H, Kurochkin AV, Pang Y, Hu W, Flynn GC &
Zuiderweg ER (1998) NMR solution structure of the
21 kDa chaperone protein DnaK substrate binding
domain: a preview of chaperone–protein interaction.
Biochemistry 37, 7929–7940.
7 Bukau B & Horwich AL (1998) The Hsp70 and Hsp60
chaperone machines. Cell 92, 351–366.
8 Blond-Elguindi S, Cwirla SE, Dower WJ, Lipshutz RJ,
Sprang SR, Sambrook JF & Gething MJ (1993) Affinity
panning of a library of peptides displayed on bacterio-
phages reveals the binding specificity of BiP. Cell 75,
717–728.
9 Gragerov A & Gottesman ME (1994) Different peptide
binding specificities of hsp70 family members. J Mol
Biol 241, 133–135.
10 James P, Pfund C & Craig EA (1997) Functional speci-
ficity among Hsp70 molecular chaperones. Science 275,
387–389.
11 Mayer MP & Bukau B (1998) Hsp70 chaperone sys-
tems: diversity of cellular functions and mechanism of

action. Biol Chem 379, 261–268.
12 Boorstein WR, Ziegelhoffer T & Craig EA (1994) Mole-
cular evolution of the HSP70 multigene family. J Mol
Evol 38 , 1–17.
13 Karlin S & Brocchieri L (1998) Heat shock protein 70
family: multiple sequence comparisons, function, and
evolution. J Mol Evol 47, 565–577.
14 Liberek K, Marszalek J, Ang D, Georgopoulos C &
Zylicz M (1991) Escherichia coli DnaJ and GrpE heat
Hsp70 structure and specificity F. Moro et al.
3194 FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS
shock proteins jointly stimulate ATPase activity of
DnaK. Proc Natl Acad Sci USA 88, 2874–2878.
15 Rowley N, Prip-Buus C, Westermann B, Brown C,
Schwarz E, Barrell B & Neupert W (1994) Mdj1p, a
novel chaperone of the DnaJ family, is involved in
mitochondrial biogenesis and protein folding. Cell 77,
249–259.
16 Bolliger L, Deloche O, Glick BS, Georgopoulos C, Jeno
P, Kronidou N, Horst M, Morishima N & Schatz G
(1994) A mitochondrial homolog of bacterial GrpE
interacts with mitochondrial hsp70 and is essential for
viability. EMBO J 13, 1998–2006.
17 Horst M, Oppliger W, Rospert S, Schonfeld HJ, Schatz
G & Azem A (1997) Sequential action of two hsp70
complexes during protein import into mitochondria.
EMBO J 16, 1842–1849.
18 Moro F, Okamoto K, Donzeau M, Neupert W &
Brunner M (2002) Mitochondrial protein import:
molecular basis of the ATP-dependent interaction of

MtHsp70 with Tim44. J Biol Chem 277, 6874–
6880.
19 Deloche O, Kelley WL & Georgopoulos C (1997) Struc-
ture–function analyses of the Ssc1p, Mdj1p, and Mge1p
Saccharomyces cerevisiae mitochondrial proteins in
Escherichia coli. J Bacteriol 179, 6066–6075.
20 Milisav I, Moro F, Neupert W & Brunner M (2001)
Modular structure of the TIM23 preprotein translocase
of mitochondria. J Biol Chem 276, 25856–25861.
21 Schneider HC, Berthold J, Bauer MF, Dietmeier K,
Guiard B, Brunner M & Neupert W (1994) Mitochon-
drial Hsp70 ⁄ MIM44 complex facilitates protein import.
Nature 371, 768–774.
22 Zhang XP, Elofsson A, Andreu D & Glaser E (1999)
Interaction of mitochondrial presequences with
DnaK and mitochondrial hsp70. J Mol Biol 288, 177–
190.
23 Bukau B & Walker GC (1989) Delta dnaK52 mutants
of Escherichia coli have defects in chromosome segrega-
tion and plasmid maintenance at normal growth tem-
peratures. J Bacteriol 171, 6030–6038.
24 Gragerov A, Zeng L, Zhao X, Burkholder W & Gottes-
man ME (1994) Specificity of DnaK-peptide binding.
J Mol Biol 235, 848–854.
25 Okamoto K, Brinker A, Paschen SA, Moarefi I, Hayer-
Hartl M, Neupert W & Brunner M (2002) The protein
import motor of mitochondria: a targeted molecular
ratchet driving unfolding and translocation. EMBO J
21, 3659–3671.
26 Azem A, Oppliger W, Lustig A, Jeno P, Feifel B,

Schatz G & Horst M (1997) The mitochondrial hsp70
chaperone system. Effect of adenine nucleotides, peptide
substrate, and mGrpE on the oligomeric state of
mhsp70. J Biol Chem 272, 20901–20906.
27 Montgomery DL, Morimoto RI & Gierasch LM (1999)
Mutations in the substrate binding domain of the
Escherichia coli 70 kDa molecular chaperone, DnaK,
which alter substrate affinity or interdomain coupling.
J Mol Biol 286, 915–932.
28 Zmijewski MA, Kwiatkowska JM & Lipinska B (2004)
Complementation studies of the DnaK-DnaJ-GrpE
chaperone machineries from Vibrio harveyi and Escheri-
chia coli, both in vivo and in vitro. Arch Microbiol 182,
436–449.
29 Pierpaoli EV, Sandmeier E, Schonfeld HJ & Christen P
(1998) Control of the DnaK chaperone cycle by substoi-
chiometric concentrations of the co-chaperones DnaJ
and GrpE. J Biol Chem 273, 6643–6649.
30 Kubo Y, Tsunehiro T, Nishikawa S, Nakai M, Ikeda E,
Toh-e A, Morishima N, Shibata T & Endo T (1999)
Two distinct mechanisms operate in the reactivation
of heat-denatured proteins by the mitochondrial
Hsp70 ⁄ Mdj1p ⁄ Yge1p chaperone system. J Mol Biol
286, 447–464.
31 Buczynski G, Slepenkov SV, Sehorn MG & Witt SN
(2001) Characterization of a lidless form of the mole-
cular chaperone DnaK: deletion of the lid increases
peptide on- and off-rate constants. J Biol Chem 276,
27231–27236.
32 Moro F, Fernandez-Saiz V & Muga A (2004) The lid

subdomain of DnaK is required for the stabilization of
the substrate-binding site. J Biol Chem 279, 19600–
19606.
33 Krzewska J, Langer T & Liberek K (2001) Mitochon-
drial Hsp78, a member of the Clp ⁄ Hsp100 family in
Saccharomyces cerevisiae, cooperates with Hsp70 in pro-
tein refolding. FEBS Lett 489, 92–96.
34 Banecki B, Zylicz M, Bertoli E & Tanfani F (1992)
Structural and functional relationships in DnaK and
DnaK756 heat-shock proteins from Escherichia coli.
J Biol Chem 267 , 25051–25058.
35 Weiss C, Niv A & Azem A (2002) Two-step purification
of mitochondrial Hsp70, Ssc1p, using Mge1p (His)(6)
immobilized on Ni-agarose. Protein Expr Purif 24,
268–273.
36 Moro F, Fernandez V & Muga A (2003) Interdomain
interaction through helices A and B of DnaK peptide
binding domain. FEBS Lett 533, 119–123.
37 Buchberger A, Theyssen H, Schroder H, McCarty JS,
Virgallita G, Milkereit P, Reinstein J & Bukau B (1995)
Nucleotide-induced conformational changes in the
ATPase and substrate binding domains of the DnaK
chaperone provide evidence for interdomain communi-
cation. J Biol Chem 270, 16903–16910.
38 Kamath-Loeb AS, Lu CZ, Suh WC, Lonetto MA &
Gross CA (1995) Analysis of three DnaK mutant pro-
teins suggests that progression through the ATPase
cycle requires conformational changes. J Biol Chem 270,
30051–30059.
39 Freeman BC, Myers MP, Schumacher R & Morimoto

RI (1995) Identification of a regulatory motif in Hsp70
F. Moro et al. Hsp70 structure and specificity
FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS 3195
that affects ATPase activity, substrate binding and inter-
action with HDJ-1. EMBO J 14, 2281–2292.
40 Kassenbrock CK & Kelly RB (1989) Interaction of
heavy chain binding protein (BiP ⁄ GRP78) with adenine
nucleotides. EMBO J 8, 1461–1467.
41 von Ahsen O, Voos W, Henninger H & Pfanner N
(1995) The mitochondrial protein import machinery.
Role of ATP in dissociation of the Hsp70.Mim44 com-
plex. J Biol Chem 270, 29848–29853.
42 Montgomery D, Jordan R, McMacken R & Freire E
(1993) Thermodynamic and structural analysis of the
folding ⁄ unfolding transitions of the Escherichia coli
molecular chaperone DnaK. J Mol Biol 232, 680–692.
43 Brodsky JL, Hamamoto S, Feldheim D & Schekman R
(1993) Reconstitution of protein translocation from
solubilized yeast membranes reveals topologically dis-
tinct roles for BiP and cytosolic Hsc70. J Cell Biol 120,
95–102.
44 Wiech H, Buchner J, Zimmermann M, Zimmermann R
& Jakob U (1993) Hsc70, immunoglobulin heavy chain
binding protein, and Hsp90 differ in their ability to sti-
mulate transport of precursor proteins into mammalian
microsomes. J Biol Chem 268, 7414–7421.
45 Suppini JP, Amor M, Alix JH & Ladjimi MM (2004)
Complementation of an Escherichia coli DnaK defect by
Hsc70–DnaK chimeric proteins. J Bacteriol 186, 6248–
6253.

46 Rassow J, Voos W & Pfanner N (1995) Partner proteins
determine multiple functions of Hsp70. Trends Cell Biol
5, 207–212.
47 Chesnokova LS, Slepenkov SV, Protasevich II,
Sehorn MG, Brouillette CG & Witt SN (2003) Deletion
of DnaK’s lid strengthens binding to the nucleotide
exchange factor, GrpE: a kinetic and thermodynamic
analysis. Biochemistry 42, 9028–9040.
48 Greene MK, Maskos K & Landry SJ (1998) Role of the
J-domain in the cooperation of Hsp40 with Hsp70. Proc
Natl Acad Sci USA 95 , 6108–6113.
49 Suh WC, Burkholder WF, Lu CZ, Zhao X, Gottesman
ME & Gross CA (1998) Interaction of the Hsp70 mole-
cular chaperone, DnaK, with its cochaperone DnaJ.
Proc Natl Acad Sci U S A 95, 15223–15228.
50 Pellecchia M, Montgomery DL, Stevens SY, Vander
Kooi CW, Feng HP, Gierasch LM & Zuiderweg ER
(2000) Structural insights into substrate binding by the
molecular chaperone DnaK. Nat Struct Biol 7, 298–303.
51 Chou CC, Forouhar F, Yeh YH, Shr HL, Wang C &
Hsiao CD (2003) Crystal structure of the C-terminal
10-kDa subdomain of Hsc70. J Biol Chem 278, 30311–
30316.
52 Zylicz M, Yamamoto T, McKittrick N, Sell S & Geor-
gopoulos C (1985) Purification and properties of the
dnaJ replication protein of Escherichia coli. J Biol Chem
260, 7591–7598.
53 Mehl AF, Heskett LD & Neal KM (2001) A GrpE
mutant containing the NH(2)-terminal ‘tail’ region is
able to displace bound polypeptide substrate from

DnaK. Biochem Biophys Res Commun 282, 562–569.
54 Lakowicz JR (1999) Principles of Fluorescence Spectros-
copy, 2nd edn. Kluwer Academic, New York.
3196 FEBS Journal 272 (2005) 3184–3196 ª 2005 FEBS
Hsp70 structure and specificity F. Moro et al.