Structure-Function Analysis of HscC, theEscherichia coli Member of a Novel Subfamily of Specialized Hsp70 Chaperones
2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês
10.1074/jbc.m206520200
ISSN1083-351X
AutoresChristoph J. Kluck, Holger Patzelt, Pierre Genevaux, Dirk Brehmer, Wolfgang Rist, Jens Schneider‐Mergener, Bernd Bukau, Matthias P. Mayer,
Tópico(s)Protein Structure and Dynamics
ResumoHsp70 chaperones assist protein folding processes through nucleotide-controlled cycles of substrate binding and release. In our effort to understand the structure-function relationship within the Hsp70 family of proteins, we characterized the Escherichia coli member of a novel Hsp70 subfamily, HscC, and identified considerable differences to the well studied E. colihomologue, DnaK, which together suggest that HscC is a specialized chaperone. The basal ATPase cycle of HscC hadk cat and K m values that were 8- and 10,000-fold higher than for DnaK. The HscC ATPase was not affected by the nucleotide exchange factor of DnaK GrpE and stimulated 8-fold by DjlC, a DnaJ protein with a putative transmembrane domain, but not by other DnaJ proteins tested. Substrate binding dynamics and substrate specificity differed significantly between HscC and DnaK. These differences are explicable by distinct structural variations. HscC does not have general chaperone activity because it did not assist refolding of a denatured model substrate. In vivo, HscC failed to complement temperature sensitivity of ΔdnaKcells. Deletion of hscC caused a slow growth phenotype that was suppressed after several generations. Triple knock-outs of allE. coli genes encoding Hsp70 proteins (ΔdnaKΔhscA ΔhscC) were viable, indicating that Hsp70 proteins are not strictly essential for viability. An extensive search for ΔhscC phenotypes revealed a hypersensitivity to Cd2+ ions and UV irradiation, suggesting roles of HscC in the cellular response to these stress treatments. Together our data show that the Hsp70 structure exhibits an astonishing degree of adaptive variations to accommodate requirements of a specialized function. Hsp70 chaperones assist protein folding processes through nucleotide-controlled cycles of substrate binding and release. In our effort to understand the structure-function relationship within the Hsp70 family of proteins, we characterized the Escherichia coli member of a novel Hsp70 subfamily, HscC, and identified considerable differences to the well studied E. colihomologue, DnaK, which together suggest that HscC is a specialized chaperone. The basal ATPase cycle of HscC hadk cat and K m values that were 8- and 10,000-fold higher than for DnaK. The HscC ATPase was not affected by the nucleotide exchange factor of DnaK GrpE and stimulated 8-fold by DjlC, a DnaJ protein with a putative transmembrane domain, but not by other DnaJ proteins tested. Substrate binding dynamics and substrate specificity differed significantly between HscC and DnaK. These differences are explicable by distinct structural variations. HscC does not have general chaperone activity because it did not assist refolding of a denatured model substrate. In vivo, HscC failed to complement temperature sensitivity of ΔdnaKcells. Deletion of hscC caused a slow growth phenotype that was suppressed after several generations. Triple knock-outs of allE. coli genes encoding Hsp70 proteins (ΔdnaKΔhscA ΔhscC) were viable, indicating that Hsp70 proteins are not strictly essential for viability. An extensive search for ΔhscC phenotypes revealed a hypersensitivity to Cd2+ ions and UV irradiation, suggesting roles of HscC in the cellular response to these stress treatments. Together our data show that the Hsp70 structure exhibits an astonishing degree of adaptive variations to accommodate requirements of a specialized function. The 70-kDa heat shock proteins (Hsp70s) 1The abbreviations used are: Hsp70, 70-kDa heat shock protein; IPTG, isopropyl-1-thio-β-d-galactopyranoside; IAANS, 2-(4′-(iodoacetamido)-anilino)naphthalene-6-sulfonic acid. 1The abbreviations used are: Hsp70, 70-kDa heat shock protein; IPTG, isopropyl-1-thio-β-d-galactopyranoside; IAANS, 2-(4′-(iodoacetamido)-anilino)naphthalene-6-sulfonic acid. constitute a large family of highly conserved chaperones that assist a multitude of protein folding processes including de novo folding of proteins, prevention of aggregation and refolding of stress denatured proteins, disaggregation of protein aggregates, control of activity and stability of regulatory proteins, and degradation of unfolded proteins (1Georgopoulos C. Liberek K. Zylicz M. Ang D. Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 209-250Google Scholar, 2Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Google Scholar, 3Mayer M.P. Brehmer D. Gässler C.S. Bukau B. Horwich A.L. Advances in Protein Chemistry: Protein Folding in the Cell. 59. Academic Press, San Diego2001: 1-44Google Scholar). Such a large and diverse array of functions has been attributed to no other class of chaperones. The chaperone activity of the Hsp70 proteins is based on several properties: (i) The C-terminal substrate-binding domain of Hsp70s interacts with substrates by binding to short peptide stretches of ∼5 amino acids in length (4Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M. Hendrickson W.A. Science. 1996; 272: 1606-1614Google Scholar). (ii) This interaction is controlled by the nucleotide status of the adjacent N-terminal ATPase domain (5Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Google Scholar). (iii) The nucleotide status is regulated by co-chaperones of the DnaJ protein family and a nucleotide exchange factor (GrpE in bacteria, Bag in eukaryotic cytosol) (3Mayer M.P. Brehmer D. Gässler C.S. Bukau B. Horwich A.L. Advances in Protein Chemistry: Protein Folding in the Cell. 59. Academic Press, San Diego2001: 1-44Google Scholar, 6Kelley W.L. Curr. Biol. 1999; 9: R305-R308Google Scholar). This mode of action of Hsp70 chaperones and their co-chaperones has been elucidated mainly using Escherichia coli DnaK with DnaJ and GrpE and mammalian Hsc70 with Hdj-1, the human DnaJ homologue, and Bag-1 M as model systems. However, more recently it was found that there are significant differences between the Hsp70 systems. In the yeast cytosol Ssa1 and Ssb1 and in E. coli DnaK and HscA (Hsc66) differ greatly with respect to their ATPase cycle. HscA and Ssb1 have much lower affinities for ATP than DnaK and Ssa1 (7Lopez-Buesa P. Pfund C. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15253-15258Google Scholar, 8Silberg J.J. Vickery L.E. J. Biol. Chem. 2000; 275: 7779-7786Google Scholar, 9Brehmer D. Rüdiger S. Gässler C.S. Klostermeier D. Packschies L. Reinstein J. Mayer M.P. Bukau B. Nat. Struct. Biol. 2001; 8: 427-432Google Scholar). These differences go along with differences in the cellular functions of these proteins, although it is not clear whether a causal correlation exists. Although Ssa1 is involved in a large variety of protein folding processes, Ssb1 seems to be mainly engaged in the translation process and the de novo folding of proteins (10Craig E. Yan W. James P. Bukau B. Molecular Chaperones and Folding Catalysts: Regulation, Cellular Function and Mechanisms. Harwood Academic Publishers, Amsterdam, The Netherlands1999: 139-162Google Scholar, 11Pfund C. Lopez-Hoyo N. Ziegelhoffer T. Schilke B.A. Lopez-Buesa P. Walter W.A. Wiedmann M. Craig E.A. EMBO J. 1998; 17: 3981-3989Google Scholar). Similarly, whereas DnaK is involved in many different folding processes in E. coli, HscA seems to be specialized for the assembly of iron-sulfur cluster proteins (12Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Google Scholar). Sequencing of the E. coli genome identified a third Hsp70 homologue, termed HscC (Hsc62). HscC shares approximately 30% amino acid identity with DnaK and HscA, which is much lower than the sequence identity between DnaK and human Hsc70 of approximately 50% (13Itoh T. Matsuda H. Mori H. DNA Res. 1999; 6: 299-305Google Scholar). HscC thus belongs to the most distant Hsp70 relatives of DnaK and is therefore anticipated to exhibit strong differences to DnaK in functional and mechanistic features. In an effort to dissect the molecular basis for the functional variability among Hsp70 chaperones and to understand the roles for this newly identified third Hsp70 homologue of E. coli, we characterized the HscC proteinin vitro and searched for its function in vivo. Routinely, the bacterial strains listed in Table I were cultured at 30 °C in NZY medium (10 g·liter−1 NZ-amine (Interorgana Chemiehandel, Köln, Germany), 5 g·liter−1 yeast extract, 5 g·l−1 NaCl). The antibiotics ampicillin, chloramphenicol, kanamycin, spectinomycin, and tetracycline (Sigma) were used at final concentrations of 100, 15, 25, 50, and 10 μg·ml−1, respectively. λ plaque formation was tested using λvir and, as control, a dnaK anddnaJ transducing λ phage (a generous gift from C. Georgopoulos).Table IBacterial strainsStrainRelevant genetic markerSourceMC4100dnaK + hscA + hscC + djlB + djlC +ATCCC600dnaK + hscA + hscC + djlB + djlC +Laboratory collectionW3100dnaK + hscA + hscC + djlB + djlC +ATCCMG1655dnaK + hscA + hscC + djlB + djlC +ATCCDH5αdnaK + hscA + hscC + djlB + djlC +Laboratory collectionB178dnaK + hscA + hscC + djlB + djlC +Laboratory collectionBB1553MC4100 ΔdnaK52∷cat sidB1Ref. 45Bukau B. Walker G. EMBO J. 1990; 9: 4027-4036Google ScholarBB4513MC4100 hscA∷cat zfh-208∷Tn10Ref. 43Hesterkamp T. Bukau B. EMBO J. 1998; 17: 4818-4828Google ScholarBB4514BB1553 zfh-208∷Tn10Ref.43Hesterkamp T. Bukau B. EMBO J. 1998; 17: 4818-4828Google ScholarBB4515MC4100zfh-208∷Tn10Ref. 43Hesterkamp T. Bukau B. EMBO J. 1998; 17: 4818-4828Google ScholarBB4516BB1553 hscA∷cat zfh-208∷Tn10Ref. 43Hesterkamp T. Bukau B. EMBO J. 1998; 17: 4818-4828Google ScholarBB5104MC4100 ΔhscC∷Sp RThis studyBB5105MC4100 ΔhscC∷KmRcrcA280∷Tn10This studyBB5106BB4515 ΔhscC∷KmRcrcA280∷Tn10This studyBB5107BB4513 ΔhscC∷KmRcrcA280∷Tn10This studyBB5108BB4514 ΔhscC∷KmRcrcA280∷Tn10This studyBB5109BB4516 ΔhscC∷KmRcrcA280∷Tn10This studyBB5110MC4100 ΔdjlB∷KmRΔdjlC∷catThis study Open table in a new tab The open reading frame encoding for HscC was PCR-amplified from MC4100 genomic DNA and cloned into the vector pUHE21–2fdΔ12 using BamHI and HindIII restriction sites. The vector contains a strong IPTG-regulatable promoter. HscC was produced in ΔdnaK52mutant cells (BB1553) and purified as follows. The hscCexpressing strain was grown in 2× YT at 30 °C to mid-log phase and induced by the addition of IPTG to a final concentration of 1 mm for 5 h. The cells were disrupted using a French Press, and cell debris was removed by centrifugation. The cleared lysate was subjected to ammonium sulfate precipitation at 35% saturation. The pellet was resuspended in a minimal volume of buffer A (25 mm Hepes·KOH, pH 7.6, 50 mm KCl, 5 mm MgCl2, 10 mmβ-mercaptoethanol, 1 mm EDTA, 5% glycerol, and 10 mm N-octylglucopyranosid) and chromatographed on a Superdex 200 gel filtration column (Amersham Biosciences) pre-equilibrated in buffer A. HscC-containing fractions were pooled and applied onto a DMAE-Fractogel anion exchange column (Merck) equilibrated in buffer A. HscC was eluted using a linear gradient of 50–500 mm KCl. The pooled HscC containing fractions were divided in aliquots and stored at −80 °C. To produce DjlCΔTM as a C-terminal fusion with intein and chitin-binding protein, the DjlCΔTM encoding DNA sequence was amplified by PCR and cloned into vector pTYB2 (New England Biolabs). DjlCΔTM-intein-chitin-binding protein was produced at 15 °C in BL21(DE3) cells grown in 2× YT medium and purified in the presence of 0.2% Triton X-100 on a chitin column according to the instructions of the manufacturer (New England Biolabs). Cleavage of the intein portion was performed in 50 mm dithiothreitol for 36 h at 4 °C. DnaK, DnaJ, CbpA, HscB, and GrpE were expressed in E. coli and purified according to published protocols (14Buchberger A. Schröder H. Büttner M. Valencia A. Bukau B. Nat. Struct. Biol. 1994; 1: 95-101Google Scholar, 15Schönfeld H.-J. Schmidt D. Zulauf M. Prog. Colloid Polym. Sci. 1995; 99: 7-10Google Scholar, 16Packschies L. Theyssen H. Buchberger A. Bukau B. Goody R.S. Reinstein J. Biochemistry. 1997; 36: 3417-3422Google Scholar, 17Ueguchi C. Kakeda M. Yamada H. Mizuno T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1054-1058Google Scholar, 18Silberg J.J. Hoff K.G. Vickery L.E. J. Bacteriol. 1998; 180: 6617-6624Google Scholar). Peptide binding to HscC was determined using a IAANS-labeled peptide (ς32-Q132-Q144-C) as published (19McCarty J.S. Rüdiger S. Schönfeld H.-J. Schneider-Mergener J. Nakahigashi K. Yura T. Bukau B. J. Mol. Biol. 1996; 256: 829-837Google Scholar). Peptide library scanning was performed essentially as described (20Rüdiger S. Germeroth L. Schneider-Mergener J. Bukau B. EMBO J. 1997; 16: 1501-1507Google Scholar). Briefly, DnaK (0.2 μm) and HscC (0.8 μm) were incubated with cellulose membranes onto which 13-mer peptides scanning the sequences of EF-Tu, MetE, and GlnRS were synthesized by peptide spot synthesis (557 peptides total). The membranes were washed, and bound protein was transferred by fractionated electroblotting onto polyvinylidene difluoride membranes for immunodetection using the ECFTM detection kit (Amersham Biosciences). Quantification of all spots using MacBas software (v2.5 Fuji) resulted in values corresponding to the affinity of the peptides to DnaK and HscC, respectively. To determine the contribution of each amino acid to binding affinity, the peptides were sorted according to decreasing affinity (peptide 1, highest affinity; peptide 557, lowest affinity), and for each of the 20 amino acids the cumulative frequency (f X(m)) was calculated separately according to the following equation. fx(m)=1m∑i=1mNx(i)1n∑i=1nNx(i)Equation 1 where N X(i) is the total number of the amino acid X in peptide i, n is the total number of peptides in the library (557), and m is a number between 1 and n, e.g. thef A(1) is the number of alanines in peptide 1 divided by the average number of A in the library;f A(2) is the average number of alanines in peptides 1 and 2 divided by the average number of alanines in the library; f A(557) is the average occurrence of alanines in peptides 1–557 divided by the average number of alanines in the library, which is equal to 1. These cumulative frequencies (f X(m)) were plottedversus m (see Fig. 4 B), and them values between 100 and 400 were analyzed by linear regression analysis. The resulting intercepts represent the relative occurrence of each amino acid in a theoretical peptide with the characteristics of the library that has the highest affinity to DnaK and HscC, respectively. Steady-state ATP hydrolysis rates were determined as described for DnaK (21Mayer M.P. Laufen T. Paal K. McCarty J.S. Bukau B. J. Mol. Biol. 1999; 289: 1131-1144Google Scholar). The reactions were performed at 30 °C in mixtures containing buffer HKM (25 mm HEPES-KOH, pH 7.6, 50 mm KCl, 5 mm MgCl2), 0.2–0.3 μm HscC, 0.4–0.6 μm DnaJ, CbpA, HscB, DjlCΔTM, 10–1000 μm ATP, and 0.1 μCi of [α-32P]ATP (Amersham Biosciences). ATP/ADP binding and release assays were performed as described (22Theyssen H. Schuster H.-P. Bukau B. Reinstein J. J. Mol. Biol. 1996; 263: 657-670Google Scholar, 23Ha J.-H. McKay D.B. Biochemistry. 1995; 34: 11635-11644Google Scholar). Prevention of aggregation and refolding of chemically denatured Luciferase were performed essentially as described (24Schröder H. Langer T. Hartl F.-U. Bukau B. EMBO J. 1993; 12: 4137-4144Google Scholar) except that light scattering was measured at 550 nm and 25 °C. To guide our biochemical studies of HscC we first analyzed its amino acid sequence using ClustalW and Swiss Model (25Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Google Scholar, 26Peitsch M.C. BioTechnology. 1995; 13: 658-660Google Scholar, 27Peitsch M.C. Biochem. Soc. Trans. 1996; 24: 274-279Google Scholar). To search for elements that had been shown for other Hsp70 homologues to be important for the ATPase cycle and the interactions with co-chaperones and substrates, the HscC sequence was aligned with the sequences of E. coliDnaK, E. coli HscA, and human Hsc70 as representatives of the three recently identified subfamilies of the Hsp70 chaperones (9Brehmer D. Rüdiger S. Gässler C.S. Klostermeier D. Packschies L. Reinstein J. Mayer M.P. Bukau B. Nat. Struct. Biol. 2001; 8: 427-432Google Scholar) (Fig. 1, A–C). The ATPase domain of HscC is overall well conserved, including the residues, which coordinate the phosphate groups of ATP and are important for catalysis (e.g. Lys70, Glu171, Thr199; numbering according to DnaK throughout the paper unless otherwise stated; for review see Ref. 28Ha J.-H. Johnson E.R. McKay D.B. Sousa M.C. Takeda S. Wilbanks S.M. Bukau B. Molecular Chaperones and Folding Catalysts: Regulation, Cellular Function, and Mechanism. Harwood Academic Publishers, Amsterdam, The Netherlands1999: 573-607Google Scholar). However, it was surprising to discern a deletion of 19 amino acids corresponding to residues 93–111 of DnaK that otherwise exclusively occurs in Hsp70s of Gram-positive bacteria and archeans (29Gupta R.S. Golding G.B. J. Mol. Evol. 1993; 37: 573-582Google Scholar), the functional consequences of which are unknown so far (not shown). In addition, the structural elements that were identified in DnaK to be important for the tight binding of nucleotides and interaction with GrpE, including two salt bridges and an exposed loop (9Brehmer D. Rüdiger S. Gässler C.S. Klostermeier D. Packschies L. Reinstein J. Mayer M.P. Bukau B. Nat. Struct. Biol. 2001; 8: 427-432Google Scholar), are absent in HscC. HscC is therefore expected to have high nucleotide dissociation rates and to be incapable of interacting with GrpE (Fig. 1 Band data not shown). The residues in DnaK that are important for the interaction with DnaJ (Tyr145, Asn147, Asp148, Arg167, Glu217, and Val218) (30Gässler C.S. Buchberger A. Laufen T. Mayer M.P. Schröder H. Valencia A. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15229-15234Google Scholar, 31Suh W.-C. Burkholder W.F., Lu, C.Z. Zhao X. Gottesman M.E. Gross C.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15223-15228Google Scholar) are all conserved in HscC except Asn147, which is conservatively exchanged by serine. It therefore seems likely that this Hsp70 also interacts with a DnaJ protein. The substrate-binding domain (residues Val354–Pro556) is less well conserved. A homology model of residues Val354–Arg486 of HscC using Swiss Model and all available structures of the substrate-binding domain of Hsp70 proteins as template is shown in Fig.1 D. The helical lid domain (residues Gln538–Ala607 in DnaK) also seems to be present, although helices A and/or B are probably shorter by a total of four turns because of a deletion in this region (corresponding to residues 514–526 in DnaK). The substrate-interacting residues are reasonably well conserved except amino acids that form an arch over the substrate backbone in the DnaK crystal structure (Fig. 1 C) (4Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M. Hendrickson W.A. Science. 1996; 272: 1606-1614Google Scholar, 32Rüdiger S. Buchberger A. Bukau B. Nat. Struct. Biol. 1997; 4: 342-349Google Scholar). A striking difference to DnaK and Hsc70 is an insertion of four residues in the loop L1,2 and a small deletion of two amino acids in loop L5,6 leading to a displacement of the opening of the substrate-binding cavity in direction of the outer loops (Fig. 1, C and D). Together with helix B, which has probably been shortened, this could have profound influences on substrate binding by HscC compared with DnaK and most other Hsp70s, in particular because we recently showed that residues in L1,2 and L3,4 modulate substrate specificity (33Rüdiger S. Mayer M.P. Schneider-Mergener J. Bukau B. J. Mol. Biol. 2000; 304: 245-251Google Scholar). Using these characteristics in the substrate-binding domain of HscC, we performed a Blast search for homologues. Homologues with an insertion of 4 amino acids in loop L1,2 were found in the closely related enterohemeoragic E. coli, Salmonella typhimurium, and Salmonella enteritis. These Hsp70 proteins have a high degree of identity over the whole sequence and are therefore HscC orthologues. In addition, we found Hsp70 homologues with an even larger insertion of 8 or 9 residues in L1,2 inBurkholderia fungorum, Pseudomonas fluorescens, Novosphingobium aromaticivorans, Bacillus anthracis, and Ralstonia solanacearum (Fig. 1, A–C). Whether these Hsp70 homologues belong to the HscC type will be discussed below. To analyze the ATPase cycle of HscC, we first determined K m andk cat of the basal steady-state ATPase activity and found that K m and k cat of HscC were 112 ± 6 μm and 0.0046 ± 0.0002 s−1 (Fig. 2 A). Although the k cat is almost 8- and 3-fold higher than the values for DnaK and HscA, the catalytic efficiencyk cat/K m is much lower for HscC (41 m−1·s−1) than for DnaK (30,000–70,000 m−1·s−1) or even HscA (109 m−1·s−1). It is surprising that Hsp70 proteins vary that much in their basal ATPase activity. The K m for ATP is 10,000-fold higher for HscC than for DnaK and still 10-fold higher than for HscA, indicating that the affinity of HscC for nucleotide is relatively low, consistent with a high nucleotide dissociation rate as predicted above. To confirm this result, we tried to determine the dissociation rate for ADP directly using the fluorescently labeled nucleotide analogue (N8-(4-N′-methylanthraniloylaminobutyl)-8 aminoadenosine 5′-diphosphat (MABA- ADP) and indirectly using intrinsic tryptophane fluorescence (22Theyssen H. Schuster H.-P. Bukau B. Reinstein J. J. Mol. Biol. 1996; 263: 657-670Google Scholar, 23Ha J.-H. McKay D.B. Biochemistry. 1995; 34: 11635-11644Google Scholar, 34Gässler C.S. Wiederkehr T. Brehmer D. Bukau B. Mayer M.P. J. Biol. Chem. 2001; 276: 32538-32544Google Scholar). However, the measured fluorescence differences between the free and nucleotide-bound forms were too small to give reliable results (not shown). Nevertheless, circumstantial evidence suggests that the dissociation rates for nucleotides are relatively high. First, HscC (like HscA) does not bind to ATP-agarose; second, a complex of ATP with HscC cannot be isolated by rapid gel filtration. Both properties are in contrast to the behavior of DnaK and mammalian Hsp70s. The ATPase rate of HscC was not stimulated significantly by DnaJ, GrpE, or a combination of both (Fig. 2 B and data not shown), arguing that neither DnaJ nor GrpE functionally interacts with HscC. In the view of our sequence analysis, this was not surprising for GrpE (Fig.1 B). In the case of DnaJ, however, the result was unexpected, and we conclude that DnaJ is not the appropriate co-chaperone for HscC. Aside from DnaJ there are five additional DnaJ proteins in E. coli: CbpA, DjlA, HscB, YbeS, and YbeV. The unique common feature of all the DnaJ homologues is a short sequence of about 75 amino acid residues called the J-domain, which is essential for the interaction with an Hsp70 chaperone partner and for the stimulation of its ATPase activity (Fig. 1 E) (6Kelley W.L. Curr. Biol. 1999; 9: R305-R308Google Scholar, 35Laufen T. Zuber U. Buchberger A. Bukau B. Fink A.L. Goto Y. Molecular Chaperones in Proteins: Structure, function, and Mode of Action. Marcel Dekker, New York1998: 241-274Google Scholar). It has been shown genetically and/or biochemically that CbpA and DjlA interact with DnaK and HscB with HscA (18Silberg J.J. Hoff K.G. Vickery L.E. J. Bacteriol. 1998; 180: 6617-6624Google Scholar, 36Genevaux P. Wawrzynow A. Zylicz M. Georgopoulos C. Kelley W.L. J. Biol. Chem. 2001; 276: 7906-7912Google Scholar, 37Genevaux P. Schwager F. Georgopoulos C. Kelley W.L. J. Bacteriol. 2001; 183: 5747-5750Google Scholar, 38Ueguchi C. Shiozawa T. Kakeda M. Yamada H. Mizuno T. J. Bacteriol. 1995; 177: 3894-3896Google Scholar). They are therefore not likely to interact functionally with HscC. The remaining DnaJ proteins, YbeS and YbeV, which exhibit 50% amino acid identity, are encoded by two open reading frames that are in close proximity to the HscC encoding genehscC (ybeW) on the E. coli chromosome (13Itoh T. Matsuda H. Mori H. DNA Res. 1999; 6: 299-305Google Scholar). Because we show here (see below) that YbeV is a bona fide DnaJ protein, we propose to change the sequencing project names of YbeS and YbeV into DjlB and DjlC (DnaJ-like proteins Band C) in analogy to DjlA. The coding sequences of both potential co-chaperones were cloned and expressed from a regulatable promoter. Overproduction of DjlB was toxic. DjlC was very unstablein vivo, and after overproduction it was found in the insoluble membrane fraction. Both proteins are predicted to have a transmembrane domain at their C terminus. Therefore, we produced DjlC without the potential transmembrane domain (DjlCΔTM) fused C-terminally to intein and chitin-binding protein and purified it by affinity chromatography over a chitin column and dithiothreitol-induced cleavage from the intein. DjlCΔTM stimulated the ATPase activity of HscC about 8-fold and therefore seems to be the appropriate DnaJ co-chaperone for HscC (Fig. 2 B). However, it did not stimulate the ATPase activity of DnaK, nor was the J-domain of DjlC when grafted onto the C-terminal domains of DnaJ able to replace DnaJin vivo (data not shown). Therefore, DjlC is not cooperating with DnaK and is most likely specific for an interaction with HscC. Addition of GrpE did not increase the DjlC-stimulated ATPase activity of HscC (not shown). Sequence analysis and modeling have suggested that the interaction of HscC with substrates might be significantly different from the DnaK-substrate interaction. Because peptides are good model substrates for Hsp70 chaperones (39Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Google Scholar, 40McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Google Scholar), we used a fluorescently labeled peptide to analyze the HscC-substrate interaction. The peptide ς32-Q132-Q144-C-IAANS (19McCarty J.S. Rüdiger S. Schönfeld H.-J. Schneider-Mergener J. Nakahigashi K. Yura T. Bukau B. J. Mol. Biol. 1996; 256: 829-837Google Scholar) was found to bind with good affinity to HscC, and the binding resulted in an increase in fluorescence at 420 nm when excited at 335 nm. Using this peptide we determined the dissociation equilibrium constant (K d) in the absence and presence of ATP. Both values were very similar, approximately 1.5 μm (Fig.3 A and TableII). This contrasts with the affinity of this peptide to DnaK, which is about 20 times higher in the absence (K d = 0.08 μm) than in the presence of ATP (K d = 1.8 μm) (41Mayer M.P. Schröder H. Rüdiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Google Scholar). Because theK d for the peptide-HscC complex in all nucleotide states is comparable with the K d of the peptide-DnaK complex in the ATP state, we determined for ς32-Q132-Q144-C-IAANS whether the substrate-binding domain of HscC is in a constitutively open conformation with high association and dissociation rate constants (k ass and k diss) (Fig.3 B). In the absence of ATP the k dissof the peptide-HscC complex (1.4 × 10−3s−1) was similar to the k dissmeasured for the peptide-DnaK complex (0.9 × 10−3s−1) (41Mayer M.P. Schröder H. Rüdiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Google Scholar), indicating that HscC is not in an open conformation but that k ass is very low. ATP increased k diss by a factor of 19 as compared with the factor of 440–2500 determined for DnaK (39Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Google Scholar, 41Mayer M.P. Schröder H. Rüdiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Google Scholar). Therefore, ATP does influence the conformation of the substrate-binding domain of HscC, but it increases the k diss andk ass to a similar extent, thereby not influencing the equilibrium constant. We conclude that the kinetics of the HscC-substrate interaction is significantly different from the DnaK-substrate interaction.Table IIHscC interaction with a substrate peptideWithout nucleotideWith ATPFold increaseK d(μm)1.5 ± 0.61.4 ± 1.50.93k off,1(s−1)0.0014 ± 0.000020.0269 ± 0.007319.2k off,2 (s−1)0.0013 ± 0.00030.93k on(m−1 · s−1)aCalculated fromK d and k off values.9331921420.6K d values were determined by equilibrium titration of the fluorescent labeled peptide ς32-Q132-Q144-C-IAANS with HscC. The k off values were determined by following the decrease of fluorescence of the HscC-ς32-Q132-Q144-C-IAANS complex after addition of unlabeled peptide.a Calculated fromK d and k off values. Open table in a new tab K d values were determined by equilibrium titration of the fluorescent labeled peptide ς32-Q132-Q144-C-IAANS with HscC. The k off values were determined by following the decrease of fluorescence of the HscC-ς32-Q132-Q144-C-IAANS complex after addition of unlabeled peptide. Because our sequence alignment showed that residues contributing to substrate specificity in DnaK are replaced in HscC by different residues (Fig. 1 C; Met404 → Asn-Arg-Gln-Gly-Val; Ala429→ Met), we investigated the substrate specificity of HscC using the peptide library approach employed earlier for DnaK (20Rüdiger S. Germeroth L. Schneider-Mergener J. Bukau B. EMBO J. 1997; 16: 1501-1507Google Scholar) and compared HscC with DnaK. For this approach we choose peptides scanning the sequences of three E. coli proteins, EF-Tu, MetE, and GlnRS, that are thermolabile proteins and substrates of DnaK (42Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Google Scholar). The chosen proteins are large enough to give a representative number of different peptide sequences (shown for MetE in Fig.4 A). HscC showed a clear pattern of binding and nonbinding peptides. A c
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