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: 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 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 [...]... operate in the reactivation of heat-denatured proteins by the mitochondrial Hsp70 ⁄ Mdj1p ⁄ Yge1p chaperone system J Mol Biol 286, 447–464 Buczynski G, Slepenkov SV, Sehorn MG & Witt SN (2001) Characterization of a lidless form of the molecular chaperone DnaK: deletion of the lid increases peptide on- and off-rate constants J Biol Chem 276, 27231–27236 Moro F, Fernandez-Saiz V & Muga A (2004) The lid subdomain. .. Weiss and Adina Niv for advice with mtHsp70 purification, and Professor Walter Neupert for the DnaK ⁄ mtHsp70 chimeras We also thank Professor F M Goni, Dr Stefka Taneva and ˜ Dr Gorka Basanez for critically reading the manu˜ script This work was supported by the University of Basque Country (UPV 13505 ⁄ 2001) and Ministerio ´ de Educacion y Ciencia (CICYT BMC 2001 ⁄ 0950 and BFU2004-03452) FM and VF... machinery Role of ATP in dissociation of the Hsp70. Mim44 complex J Biol Chem 270, 29848–29853 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 Brodsky JL, Hamamoto S, Feldheim D & Schekman R (1993) Reconstitution of protein translocation from solubilized yeast. .. subdomain of DnaK is required for the stabilization of the substrate-binding site J Biol Chem 279, 19600– 19606 Krzewska J, Langer T & Liberek K (2001) Mitochondrial Hsp78, a member of the Clp ⁄ Hsp100 family in Saccharomyces cerevisiae, cooperates with Hsp70 in protein refolding FEBS Lett 489, 92–96 Banecki B, Zylicz M, Bertoli E & Tanfani F (1992) Structural and functional relationships in DnaK and DnaK7 56... B, Brunner M & Neupert W (1994) Mitochondrial Hsp70 ⁄ MIM44 complex facilitates protein import Nature 371, 768–774 Zhang XP, Elofsson A, Andreu D & Glaser E (1999) Interaction of mitochondrial presequences with DnaK and mitochondrial hsp70 J Mol Biol 288, 177– 190 Bukau B & Walker GC (1989) Delta dnaK5 2 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal... Kwiatkowska JM & Lipinska B (2004) Complementation studies of the DnaK- DnaJ-GrpE chaperone machineries from Vibrio harveyi and Escherichia coli, both in vivo and in vitro Arch Microbiol 182, 436–449 Pierpaoli EV, Sandmeier E, Schonfeld HJ & Christen P (1998) Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE J Biol Chem 273, 6643–6649 Kubo Y, Tsunehiro... 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 molecular 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... 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 & Georgopoulos C (1985) Purification and properties of the dnaJ replication protein of Escherichia coli J Biol Chem... molecular basis of the ATP-dependent interaction of MtHsp70 with Tim44 J Biol Chem 277, 6874– 6880 Deloche O, Kelley WL & Georgopoulos C (1997) Structure–function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli J Bacteriol 179, 6066–6075 Milisav I, Moro F, Neupert W & Brunner M (2001) Modular structure of the TIM23 preprotein translocase of mitochondria... 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 . Conformational properties of bacterial DnaK and yeast mitochondrial Hsp70 Role of the divergent C-terminal a-helical subdomain Fernando Moro 1 ,. 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