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.. Abbreviations DSC, diff
Trang 1mitochondrial Hsp70
Role of the divergent C-terminal a-helical subdomain
Fernando Moro1, Vanesa Ferna´ndez-Sa´iz1, Olga Slutsky2, Abdussalam Azem2and Arturo Muga1
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 regulatransloca-tion 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: gbbmopef@lg.ehu.es or
gbpmuvia@lg.ehu.es
(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 coliand 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 42C); (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.
Trang 2b-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 Steady-state ATPase was measured at 30 C, protein and ATP concentrations were 5 l M and 1 m M , respectively NRLLLTG (NR) peptide was added at 0.5 m M DnaJ and GrpE concentrations were 0.5 l M 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).
Trang 3Chimeric 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 42C (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)1Æmin)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 kobs 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 42C in the presence of excess protein (Fig 2B) As observed pre-viously for DnaK [32], mtHsp70 binding kinetics were
Trang 4faster 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
25C, as observed for lidless mutants [31,32] (Fig 2B), and showed a significantly reduced binding at 37C that is virtually abolished at 42C (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
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 C for 2 h
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.
Kd(l M ) k+1( M )1Æs)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
Trang 5In summary, the affinity of the wild-type proteins
and the chimeras for F-APPY was reasonably similar
at 25C 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 KSV 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).
Trang 6times; (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 (Ksv, M )1) obtained in
the absence and presence of 0.5 m M ATP Ksv 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.
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 m M KCl, 10 m M 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)1and 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 l M
Trang 7Next, 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 74C 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 51.8C 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 76.2C (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 51.6C could represent the unfold-ing of a more stable ATPase domain The Tm 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].
Trang 8interactions 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
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 subsub-strate- 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 con-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)1protein 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.
Trang 9indicates 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 42C 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 denatfail-ured
luci-ferase At stress temperatures (e.g 42C) 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-helicconformation-al 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 Tmvalues are reported in C; DH values are in kcalÆmol)1 The uncer-tainty in the experimental values is ± 0.2 C for T m and 15% for DH.
Transition 1 Transition 2 Transition 3 Transition 4
Trang 10Experimental 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 30C, 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 30C, 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 MgCl2, 1 mm 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 4C
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 42C 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 kobs 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 nm in
50 mm Tris, pH 7.7, 55 mm KCl, 15 mm MgCl2, 5.5 mm DTT, 0.5 mgÆmL)1 bovine serum albumin containing an ATP-regenerating system (4 mm phosphoenolpyruvate and
20 ngÆmL)1pyruvate 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 25C with 2 lm Hsp70 (DnaK, mtHsp70, KKCC and KCCC) which was preincubated for
15 min at 25C with 4 mm ATP in 25 mm Hepes, pH 7.5,
50 mm KCl, 5 mm MgCl2, 5 mm 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 42C Luciferase activity was measured as above after a 90 min reactivation period at 25C
Infrared spectroscopy
Proteins were extensively dialyzed against 100 mm Mops,
pH 7.0, 50 mm KCl, 10 mm MgCl2 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]