Interaction of the DnaK and DnaJ Chaperone System with a Native Substrate, P1 RepA

Abstract

DnaK, the Hsp70 chaperone of Escherichia coli interacts with protein substrates in an ATP-dependent manner, in conjunction with DnaJ and GrpE co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes. To understand how DnaJ targets specific proteins for recognition by the DnaK chaperone system, we investigated the interaction of DnaJ and DnaK with a known natural substrate, bacteriophage P1 RepA protein. By characterizing RepA deletion derivatives, we found that DnaJ interacts with a region of RepA located between amino acids 180 and 200 of the 286-amino acid protein. A peptide corresponding to amino acids 180–195 inhibited the interaction of RepA and DnaJ. Two site-directed RepA mutants with alanine substitutions in this region were about 4-fold less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA. We also identified by deletion analysis a site in RepA, in the region of amino acids 35–49, which interacts with DnaK. An alanine substitution mutant in amino acids 36–39 was constructed and found defective in activation by DnaJ and DnaK. Taken together the results suggest that DnaJ and DnaK interact with separate sites on RepA. DnaK, the Hsp70 chaperone of Escherichia coli interacts with protein substrates in an ATP-dependent manner, in conjunction with DnaJ and GrpE co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes. To understand how DnaJ targets specific proteins for recognition by the DnaK chaperone system, we investigated the interaction of DnaJ and DnaK with a known natural substrate, bacteriophage P1 RepA protein. By characterizing RepA deletion derivatives, we found that DnaJ interacts with a region of RepA located between amino acids 180 and 200 of the 286-amino acid protein. A peptide corresponding to amino acids 180–195 inhibited the interaction of RepA and DnaJ. Two site-directed RepA mutants with alanine substitutions in this region were about 4-fold less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA. We also identified by deletion analysis a site in RepA, in the region of amino acids 35–49, which interacts with DnaK. An alanine substitution mutant in amino acids 36–39 was constructed and found defective in activation by DnaJ and DnaK. Taken together the results suggest that DnaJ and DnaK interact with separate sites on RepA. DnaK, the Escherichia coli Hsp70 homologue, is an ATP-dependent molecular chaperone that acts in conjunction with the co-chaperones, DnaJ and GrpE, to mediate protein folding and remodeling reactions in the cell. The DnaK and Hsp70 chaperone systems participate in a wide variety of cellular processes in both normal and stressed cells, including nascent protein folding, protein trafficking across intracellular membranes, proteolysis, assembly of multiprotein structures, disassembly of protein aggregates, cell division, DNA replication of several phage and plasmids, and regulation of the heat shock response (see Refs. 1Frydman J. Annu. Rev. Biochem. 2001; 70: 603-647Google Scholar, 2Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Google Scholar, 3Bukau B. Deuerling E. Pfund C. Craig E.A. Cell. 2000; 101: 119-122Google Scholar for recent reviews). Structural studies have shown that DnaK, like other Hsp70s, consists of an N-terminal ATP-binding domain and a C-terminal substrate-binding domain (4Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Google Scholar, 5Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Google Scholar). Cycles of ATP binding and hydrolysis by the N-terminal domain modulate peptide binding by DnaK (6Szabo A. Langer T. Schroder H. Flanagan J. Bukau B. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10345-10349Google Scholar, 7Laufen T. Mayer M.P. Beisel C. Klostermeier D. Mogk A. Reinstein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Google Scholar, 8Mayer M.P. Rudiger S. Bukau B. Biol. Chem. 2000; 381: 877-885Google Scholar). In the ATP-bound state, DnaK binds and releases substrates rapidly, whereas in the ADP-bound state, binding and release is slow (9Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Google Scholar, 10Russell R. Wali Karzai A. Mehl A.F. McMacken R. Biochemistry. 1999; 38: 4165-4176Google Scholar). DnaJ and GrpE regulate cycling between the two states. DnaJ stimulates DnaK ATPase, forming the ADP state of DnaK, which stably interacts with the polypeptide substrate (11McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Google Scholar). In addition, DnaJ tags several proteins for recognition by DnaK (23Wickner S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2690-2694Google Scholar, 43). GrpE is a nucleotide exchange factor, binding to the ATPase domain of DnaK and inducing the exchange of bound ADP with ATP (12Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F. Kuriyan J. Science. 1997; 276: 431-435Google Scholar, 13Packschies L. Theyssen H. Buchberger A. Bukau B. Goody R.S. Reinstein J. Biochemistry. 1997; 36: 3417-3422Google Scholar). Studies to elucidate the interaction of DnaK and Hsp70 with peptides showed that the chaperones recognize heptameric extended peptides enriched in hydrophobic residues (14Flynn G.C. Pohl J. Flocco M.T. Rothman J.E. Nature. 1991; 353: 726-730Google Scholar, 15Landry S.J. Jordan R. McMacken R. Gierasch L.M. Nature. 1992; 355: 455-457Google Scholar, 16Blond-Elguindi S. Cwirla S.E. Dower W.J. Lipshutz R.J. Sprang S.R. Sambrook J.F. Gething M.J. Cell. 1993; 75: 717-728Google Scholar, 17Gragerov A. Zeng L. Zhao X. Burkholder W. Gottesman M.E. J. Mol. Biol. 1994; 235: 848-854Google Scholar). Bukau and co-workers (18Rudiger S. Germeroth L. Schneider-Mergener J. Bukau B. EMBO J. 1997; 16: 1501-1507Google Scholar) performed an extensive study of more than 4000 cellulose-bound peptides spanning sequences of biologically relevant substrates to identify a consensus DnaK binding motif. The motif consists of a hydrophobic core of four or five residues enriched in Leu and to a lesser extent in Ile, Val, Phe, and Tyr, and two flanking regions enriched in basic residues. These sites occur in proteins, not just biologically relevant proteins, on average every 36 residues (18Rudiger S. Germeroth L. Schneider-Mergener J. Bukau B. EMBO J. 1997; 16: 1501-1507Google Scholar). The DnaJ binding motif was studied by a similar method and found to consist of a hydrophobic core of about 8 residues enriched for aromatic and large aliphatic hydrophobic residues and arginine (19Rudiger S. Schneider-Mergener J. Bukau B. EMBO J. 2001; 20: 1042-1050Google Scholar). Although this motif is slightly different from the DnaK binding motif, DnaJ is able to bind most of the same peptides as DnaK (19Rudiger S. Schneider-Mergener J. Bukau B. EMBO J. 2001; 20: 1042-1050Google Scholar). Because of the hydrophobic nature of the motifs, DnaK- and DnaJ-binding sites typically reside in the interior of correctly folded proteins and are only exposed in nascent or unfolded polypeptides. Identification of the DnaJ and DnaK peptide binding motifs helps explain how the chaperone system can interact with unfolded polypeptides. However, in non-stress conditions DnaK protein comprises more than 1% of the total cell protein and is essential for normal growth (20Bukau B. Walker G.C. J. Bacteriol. 1989; 171: 2337-2346Google Scholar). DnaK would pose a danger to the cell if it indiscriminately unfolded active proteins or dissociated complexes. One of several characterized reactions that the DnaK chaperone system carries out during non-stress situations is the maintenance of plasmid mini-P1 (21Bukau B. Walker G.C. J. Bacteriol. 1989; 171: 6030-6038Google Scholar, 22Tilly K. Yarmolinsky M. J. Bacteriol. 1989; 171: 6025-6029Google Scholar). In vitro, DnaK, DnaJ, and GrpE are required for DNA replication of mini-P1 and their sole function is to activate origin-specific DNA binding by RepA by converting inactive RepA dimers to active monomers (23Wickner S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2690-2694Google Scholar, 24Wickner S. Hoskins J. McKenney K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7903-7907Google Scholar, 25Wickner S. Hoskins J. McKenney K. Nature. 1991; 350: 165-167Google Scholar, 26Skowyra D. Wickner S. J. Biol. Chem. 1993; 268: 25296-25301Google Scholar). ClpA, a member of the Clp/Hsp100 family of ATP-dependent molecular chaperones, also activates DNA binding by P1 RepA by converting inactive dimers to active monomers (27Wickner S. Gottesman S. Skowyra D. Hoskins J. McKenney K. Maurizi M.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12218-12222Google Scholar). Studies to elucidate substrate recognition by ClpA have shown that it recognizes some substrates through N-terminal motifs and others through C-terminal motifs (28Tobias J.W. Shrader T.E. Rocap G. Varshavsky A. Science. 1991; 254: 1374-1377Google Scholar, 29Gottesman S. Roche E. Zhou Y. Sauer R.T. Genes Dev. 1998; 12: 1338-1347Google Scholar, 30Hoskins J.R. Singh S.K. Maurizi M.R. Wickner S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8892-8897Google Scholar). RepA is recognized through a motif near the N terminus, between amino acids 10 and 15 (31). To address the question of how the DnaK chaperone system recognizes specific native substrates, we determined regions within a known substrate, P1 RepA, which are recognized by DnaJ and DnaK. ATP was obtained from Roche Molecular Biochemicals. Restriction endonucleases were obtained from New England BioLabs and polymerase chain reaction reagents were obtained from PerkinElmer Life Sciences. Peptides were synthesized, purified, and analyzed by mass spectrophotometry by Research Genetics Inc. Rabbit anti-DnaJ serum and mouse anti-DnaK monoclonal antibody were obtained from StressGen Biotechnologies. Rabbit anti-RepA serum was produced by Research Genetics Inc. using purified RepA. The construction of plasmids expressing N-terminal deletions of RepA, including RepA-(16–286), RepA-(26–286), RepA-(36–286), RepA-(50–286), and RepA-(1–180), was previously described (31Hoskins J.R. Kim S.Y. Wickner S. J. Biol. Chem. 2000; 275: 35361-35367Google Scholar). Plasmids expressing C-terminal deletions of RepA were constructed by generating appropriate repA polymerase chain reaction fragments containing 5′ NdeI and 3′BamHI sites. The fragments were then ligated into pET19b (Novagen), containing a N-terminal deca-His tag. The sequences of all of mutants were verified by DNA sequencing. 3H-LabeledoriP1 plasmid DNA (3590 cpm/fmol) was prepared as described (25Wickner S. Hoskins J. McKenney K. Nature. 1991; 350: 165-167Google Scholar). Site-directed alanine substitution mutants of wild type RepA-His6 were constructed using the QuikChange mutagenesis kit (Stratagene) and the protocol recommended by the supplier. In RepA-mut1, amino acids Tyr-190, Val-192, Leu-193, and Leu-194 were changed to alanines, in RepA-mut2, amino acids Leu-193, Leu-194, His-196, and His-197 were converted to alanines, and in RepA-mut3 amino acids Arg-36, Leu-37, Gly-38, and Val-39 were converted to alanines. DnaJ (32Zylicz M. Yamamoto T. McKittrick N. Sell S. Georgopoulos C. J. Biol. Chem. 1985; 260: 7591-7598Google Scholar), DnaK (26Skowyra D. Wickner S. J. Biol. Chem. 1993; 268: 25296-25301Google Scholar), GrpE (26Skowyra D. Wickner S. J. Biol. Chem. 1993; 268: 25296-25301Google Scholar), and RepA (23Wickner S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2690-2694Google Scholar) were purified as described. RepA N-terminal deletion proteins, RepA-(50–286), and alanine substitution mutants were isolated by the method described for RepA (23Wickner S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2690-2694Google Scholar). The purity of all proteins was greater than 95% as determined by SDS-PAGE. Protein concentrations are expressed as molar amounts of RepA dimers, DnaJ dimers, DnaK monomers, and GrpE dimers. Protein concentrations were determined by Bradford assay (Bio-Rad) with bovine serum albumin as a standard. Reaction mixtures (20 μl) contained Buffer A (20 mm Tris·HCl, pH 7.5, 100 mm KCl, 5 mm dithiothreitol, 0.1 mmEDTA, 10% glycerol (v/v)), 10 mm MgOAc, 1 mmATP, 100 μg/ml bovine serum albumin, 0.05 pmol of RepA, 0.5 pmol of DnaJ, and 10 pmol of DnaK, unless indicated otherwise. After 10 min at 24 °C, calf thymus DNA (1 μg) and 10 fmol of [3H]oriP1 plasmid DNA were added. The mixtures were then filtered through nitrocellulose filters, the filters were washed, and the retained radioactivity was measured. Activation by ClpA was measured in similar reaction mixtures but with 0.5 pmol of ClpA in place of DnaJ and DnaK. Reaction mixtures (50 μl) contained 1 μmRepA or the RepA derivative, and where indicated, 1 μmDnaJ, and 2 μm DnaK in Buffer A with 5 mmMgOAc, 1 mg/ml bovine serum albumin, and 1 mm ATP. After 10 min at room temperature, 400 μl of Buffer B (50 mmTris·HCl, pH 7.5, 100 mm NaCl, 0.1% Tween 20) and 1 μl of anti-RepA rabbit serum were added. The mixtures were incubated for 30 min at 4 °C with rocking. Then, 15 μl of a 1:1 (v/v) slurry of protein A-Sepharose (Amersham Biosciences) in Buffer B was added and incubated 30 min at 4 °C with rocking. The resin was collected by centrifugation and washed 4 times with 500 μl of 50 mmTris·HCl, pH 7.5, 300 mm NaCl, 0.2% Tween 20, and 0.1 mm EDTA. The precipitates were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Applied Biotechnologies) by electroblotting. Following transfer, DnaJ, DnaK, and RepA were detected with specific antibodies using a chemiluminiscence blotting substrate kit (Novex using cell expressing RepA or RepA were by a cell and then at for 30 The (50 μl) were incubated with anti-RepA rabbit serum and as RepA in 50 μl of was in by for 1 at room and then washed times with μl of with Tween DnaJ was for 10 min or with peptides in 50 μl of containing 2 mg/ml bovine serum and added to the and incubated for 10 The of bound DnaJ was determined as The were washed times with μl of and then 50 μl of containing anti-DnaJ rabbit serum was added. After 30 the were washed times with μl of and was as a were using a (Bio-Rad) and the at was measured. To the of native protein recognition by the DnaK chaperone system, we studied the interaction of DnaK and DnaJ with P1 RepA. DnaK and DnaJ activate the DNA binding of RepA by converting inactive dimers to active monomers S. Hoskins J. McKenney K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7903-7907Google Scholar, 25Wickner S. Hoskins J. McKenney K. Nature. 1991; 350: 165-167Google GrpE is also required conditions to nucleotide exchange (26Skowyra D. Wickner S. J. Biol. Chem. 1993; 268: 25296-25301Google Scholar). We N-terminal deletion mutants of RepA for their to activated by the of DnaK and DnaJ and found that RepA mutants or 15 amino acids were activated as as wild type RepA 1 A and not A deletion amino acids was also but to a lesser However, this was also defective in DNA binding activation by with that was not in chaperone recognition but in DNA recognition J.R. Kim S.Y. Wickner S. J. Biol. Chem. 2000; 275: 35361-35367Google Scholar and not of greater than amino acids were not of their DNA binding In RepA 15 N-terminal amino acids were not activated by the ClpA molecular whereas 5 or 10 amino acids were activated 1 B (31Hoskins J.R. Kim S.Y. Wickner S. J. Biol. Chem. 2000; 275: 35361-35367Google The results with the 15 and amino acids that the chaperone recognition in RepA is C-terminal to amino acid and is from the ClpA recognition motif. To the region or regions of RepA recognized by DnaJ, were to containing DnaJ and RepA or a RepA deletion were incubated with RepA and the were subjected to SDS-PAGE by analysis with DnaJ antibody as described We found that DnaJ with wild type RepA and RepA the N-terminal amino RepA-(50–286), but not with RepA the C-terminal amino and These results that a DnaJ recognition or in the C-terminal of RepA, between amino acids 180 and To the DnaJ recognition site in RepA more we constructed five RepA containing C-terminal using RepA and of expressing the were performed and RepA and DnaJ were detected by analysis of the showed that the cell contained RepA or a RepA deletion and DnaJ 2 Two of were the of We that DnaJ with RepA the C-terminal or amino acids 2 less with or amino acids 2 In DnaJ was not detected in with a amino acids These results suggest that the region in RepA between amino acids and is for the interaction with DnaJ and amino acids in the region of to the interaction with DnaJ. We performed using RepA peptides to that DnaJ recognized a site in the region of amino acids and five RepA peptides were corresponding to RepA amino acids and type RepA was in and then DnaJ or DnaJ that with of the RepA peptides was added to the After were detected by peptide inhibited the interaction of RepA and DnaJ about and peptide was inhibited about this by and peptides was A showed that 8 μm peptide inhibited the interaction about These results suggest that a DnaJ-binding site between amino acids 180 and 200 of RepA. A with the using deletion was that the proteins have and their of interaction with DnaJ not have the of a DnaJ-binding site in native RepA. to the results of the deletion analysis and peptide two site-directed mutants were constructed in the region identified by the peptide In RepA-mut1, were for Tyr-190, Val-192, Leu-193, and In the RepA-mut2, Leu-193, Leu-194, His-196, and His-197 were with alanines. We the of mutants to activated by the system 4 mutants were less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA. In ClpA activated oriP1 DNA binding by both of the mutants to a similar extent as wild that the mutant proteins are native dimers and can into active monomers 4 DNA binding by the mutant proteins was also activated to a similar extent as wild type RepA by with by not These suggest that and are defective in their to by DnaJ and DnaK and that residues for recognition are in amino acids Because peptide not to interact with DnaJ, it is that of the site are in the region of amino acids as as in the region of Taken the results of the deletion the peptide and the site-directed mutant that a DnaJ interaction site in RepA is in the region of amino acids the by of expressing C-terminal RepA with RepA in 2 were for DnaK by DnaK with all of the C-terminal deletion proteins, less to with and and In a DnaK was not detected a that not a RepA was 5 DnaK to interact with or more sites in the N-terminal amino acids of RepA. of DnaK with which not interact with DnaJ, that DnaK and DnaJ interact with separate on RepA, shown that peptides that bind DnaK also bind DnaJ (19Rudiger S. Schneider-Mergener J. Bukau B. EMBO J. 2001; 20: 1042-1050Google Scholar). To identify a region in the N-terminal of RepA recognized by DnaK, we purified N-terminal RepA deletion in the using RepA for their to with DnaK. DnaK with RepA and N-terminal amino acids 5 2 and of DnaK with the acid deletion mutant the that mutant activated for DNA binding by the DnaK chaperone system 1 In binding was between DnaK and a mutant RepA N-terminal amino RepA-(50–286), that mutant bound DnaJ 2 that is a site in RepA in the of amino acids 5 The in that region We then an alanine substitution in which Arg-36, Leu-37, Gly-38, and Val-39 were with by site-directed The mutant was not activated by DnaJ and DnaK, the of DNA binding in the and of the chaperone system was similar The mutant was activated by ClpA, that the of the mutant protein was similar to wild type in recognized by ClpA and converted to active monomers of binding DNA These results suggest that the alanine substitutions a recognition We have studied the interaction of DnaJ and DnaK with RepA to understand how the DnaK chaperone system recognizes a native protein. The to DnaJ recognition sites in RepA by peptide and site-directed identified a DnaJ-binding site in the region of amino acids acid region of RepA amino acids to found in peptides that bind DnaJ (19Rudiger S. Schneider-Mergener J. Bukau B. EMBO J. 2001; 20: 1042-1050Google Scholar). The peptide that inhibited the interaction of DnaJ and RepA to the residues to found in a DnaJ binding motif within a of amino acids The peptide amino acids in a of amino However, it inhibited binding less than the 180–195 The results that is a site in RepA between amino acids and The site in RepA, on of peptide binding by DnaK by Bukau and co-workers (18Rudiger S. Germeroth L. Schneider-Mergener J. Bukau B. EMBO J. 1997; 16: 1501-1507Google Scholar), consists of a core of five hydrophobic amino acids between residues and and several flanking basic amino Arg-36, and showed that peptides that bind DnaJ also bind DnaK (19Rudiger S. Schneider-Mergener J. Bukau B. EMBO J. 2001; 20: 1042-1050Google Scholar). The that DnaK bound RepA with a C-terminal deletion of amino acids but DnaJ not 2 B that DnaK can bind to a site or sites in RepA from DnaJ sites. The that RepA for the N-terminal amino acids bound DnaJ but not DnaK 2 5 that the DnaJ-binding site or sites in RepA are specific for DnaJ and not bound by DnaK. is that the sites identified by the deletion analysis hydrophobic regions exposed of of the deletion However, both peptide and site-directed that regions are for recognition by the DnaJ and DnaK chaperone Two have for the of substrates to DnaK by DnaJ (19Rudiger S. Schneider-Mergener J. Bukau B. EMBO J. 2001; 20: 1042-1050Google Scholar). In DnaJ binds to the substrate through specific DnaJ-binding sites. By the the and sites on the substrate are and the of to the substrate is with the of DnaJ from the substrate and the of DnaJ with DnaK, in a reaction and ATP hydrolysis by DnaK. In the DnaK with a different site on the substrate than DnaJ, generating a by and and ATP hydrolysis by DnaK. suggest that by GrpE, results in in DnaK that release of the the in which RepA the DnaK chaperone system by and with DnaJ and DnaK. The of known natural substrates for the DnaK chaperone system is and their interaction with DnaJ and DnaK not characterized The DNA replication of bacteriophage is by the DnaK chaperone system in a reaction the release of and some and in the activation of and DNA replication C. McMacken R. J. Biol. Chem. 1989; Scholar). Studies with purified proteins have that both and interact with DnaJ and DnaK C. McMacken R. J. Biol. Chem. 1989; Scholar, A. M. J. Biol. Chem. 1995; Scholar, A. B. D. K. Georgopoulos C. M. J. Biol. Chem. 1995; Scholar). The proteins of plasmids and are activated and by DnaJ and DnaK, by a similar to P1 RepA M. C. Y. T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: K. J. Biol. Chem. Scholar). In the of the the reaction an molecular K. J. Biol. Chem. Scholar). The DnaK chaperone system also acts on the heat shock factor, to D. W. Genes Dev. 1990; Scholar) and in interacts with both DnaJ and DnaK A. M. J. Biol. Chem. 1995; Scholar, J. H. Bukau B. Cell. 1992; Scholar). is to DnaK and DnaJ recognize separate sites on specific substrates as by the with RepA.