Scanning Mutagenesis Identifies Amino Acid Residues Essential for the in Vivo Activity of the Escherichia coli DnaJ (Hsp40) J-Domain

Corresponding author: Pierre Genevaux, Centre Médical Universitaire, 1, rue Michel-Servet, CH-1211 Genève 4, Switzerland., pierre.genevaux{at}medecine.unige.ch (E-mail)

Communicating editor: A. L. SONENSHEIN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The DnaJ (Hsp40) cochaperone regulates the DnaK (Hsp70) chaperone by accelerating ATP hydrolysis in a cycle closely linked to substrate binding and release. The J-domain, the signature motif of the Hsp40 family, orchestrates interaction with the DnaK ATPase domain. We studied the J-domain by creating 42 mutant E. coli DnaJ variants and examining their phenotypes in various separate in vivo assays, namely, bacterial growth at low and high temperatures, motility, and propagation of bacteriophage . Most mutants studied behaved like wild type in all assays. In addition to the ₃₃HisProAsp₃₅ (HPD) tripeptide found in all known functional J-domains, our study uncovered three new single substitution mutations (Y25A, K26A, and F47A) that totally abolish J-domain function. Furthermore, two glycine substitution mutants in an exposed flexible loop (R36G, N37G) showed partial loss of J-domain function alone and complete loss of function as a triple (RNQ-GGG) mutant coupled with the phenotypically silent Q38G. Interestingly, all the essential residues map to a small region on the same solvent-exposed face of the J-domain. Engineered mutations in the corresponding residues of the human Hdj1 J-domain grafted in E. coli DnaJ also resulted in loss of function, suggesting an evolutionarily conserved interaction surface. We propose that these clustered residues impart critical sequence determinants necessary for J-domain catalytic activity and reversible contact interface with the DnaK ATPase domain.

THE Hsp70 chaperone machine performs many diverse roles in the cell, including folding of nascent proteins, translocation of polypeptides across organelle membranes, coordinating responses to stress, and targeting selected proteins for degradation (HARTL 1996 ; BUKAU and HORWICH 1998 ). Hsp70 chaperone machines can participate in more specialized roles, such as signaling and vesicle trafficking, and such activities have been exploited by viruses, such as influenza, SV40, and bacteriophage (SULLIVAN et al. 2000 ). Mechanistically, the Hsp70 chaperone couples ATP hydrolysis in its N-terminal domain to substrate binding and release in its C-terminal domain. The Hsp40 cochaperones specifically help stimulate ATP hydrolysis and deliver substrates to Hsp70, while other factors, such as GrpE and Bag1, participate in nucleotide exchange (HARTL 1996 ; GASSLER et al. 2001 ).

The J-domain represents an 70-amino-acid-long residue signature sequence of the Hsp40 family and helps direct specific interaction with the Hsp70 ATPase domain (LIBEREK et al. 1991 ; WALL et al. 1994 ; KARZAI and MCMACKEN 1996 ; LYMAN and SCHEKMAN 1997 ; GASSLER et al. 1998 ; GREENE et al. 1998 ; KELLEY 1998 ; MISSELWITZ et al. 1998 ; SUH et al. 1998 ). Multiple alignments of various J-domains show significant overall amino acid sequence conservation among evolutionarily diverse organisms (HENNESSY et al. 2000 ). High-resolution NMR showed that the J-domain of the Escherichia coli DnaJ protein represents a novel fold, composed of two antiparallel coiled coil helices flanking a flexibly disordered solvent-exposed loop that contains the highly conserved His-Pro-Asp (HPD) tripeptide (SZYPERSKI et al. 1994 ; HILL et al. 1995 ; PELLACCHIA et al. 1996 ; HUANG et al. 1999 ). The structures of the J-domains of the human Hdj1 (QIAN et al. 1996 ), the polyomavirus T antigen (BERJANSKII et al. 2000 ), and the SV40 T antigen in complex with the retinoblastoma tumor-suppressor pocket domain (KIM et al. 2001 ) show a similar overall domain fold, implying an evolutionarily conserved mechanism of Hsp40-Hsp70 interaction. Amino acid substitutions in the HPD tripeptide in the E. coli DnaJ, as well as in other Hsp40 J-domains, abolish stimulation of their cognate Hsp70 partner ATPase activity (WALL et al. 1994 ; TSAI and DOUGLAS 1996 ) and result in loss of function in vivo.

In this study, we sought to identify amino acid side chains that comprise the regulatory DnaK/DnaJ contact interface. Alanine and glycine scanning mutants across the entire J-domain coding sequence in E. coli DnaJ were engineered, and their mutant phenotypes were analyzed in vivo using several independent assays for functional DnaJ cochaperone activity, namely, bacterial growth at low and high temperature (WALL et al. 1994 ; UEGUCHI et al. 1995 ), motility (SHI et al. 1992 ), and bacteriophage growth. This genetic approach has allowed the characterization of novel amino acid residues critical for J-domain biological function.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Bacterial strains and plasmids:
E. coli WKG190 is MC4100 dnaJ::Tn10-42 cbpA::kan and has been described previously (KELLEY and GEORGOPOULOS 1997 ). MG1655 dnaJ::Tn10 cbpA::kan was constructed by standard bacteriophage P1 transduction. Plasmids pWKG90 and pWKG90H71T have been described (KELLEY and GEORGOPOULOS 1997 ). A 210-bp fragment encoding the J-domain of Hdj1 (RAABE and MANLEY 1991 ) was amplified from human genomic DNA (Stratagene, La Jolla, CA) by the polymerase chain reaction (PCR) using the primers 5'-cggaattccatgggtaaagactactaccagacgttg-3' and 5'-ccggtaccttcctccccgtagcggtcgaagatctc-3', which introduced the appropriate EcoRI and KpnI sites (underlined). The fragment was digested with EcoRI and KpnI and cloned into plasmid pWKG90H71T digested with the same enzymes to yield pGP18. All DNA manipulations were performed with DH5 or DH10B (Invitrogen/Life Technologies).

Mutagenesis:
To be consistent with the DnaJ numbering in the Protein Database (PDB) accession 1XBL (PELLACCHIA et al. 1996 ), the initiating methionine codon is taken as 1. The dnaJ mutants were constructed by two-step PCR site-directed mutagenesis (HIGUCHI et al. 1988 ), by Quickchange (Stratagene), or by primer extension (KUNKEL et al. 1987 ) using plasmid pWKG90 as template. The deletion mutant dnaJ helixIV was constructed using the primer L57 + Kpn 5'-ccggtacccagaacttcataagcttc-3' (KpnI site underlined) and the upstream fusion PCR primer followed by EcoRI-KpnI digestion and subcloning into pWKG90H71T cleaved with the same enzymes. The numbering for all hdj1 mutants uses methionine start codon as 0, consistent with PDB accession 1HDJ (QIAN et al. 1996 ). Hdj1 J-domain mutants were constructed in the chimeric plasmid pGP18, using the same two-step PCR-directed mutagenesis strategy. All PCR reactions were performed using Pfu polymerase (Stratagene), and the constructs were sequence verified using the appropriate primers.

Immunoblot analysis:
Whole-cell extracts were prepared as previously described (KELLEY and GEORGOPOULOS 1997 ). For steady-state protein analysis, DnaJ proteins were resolved in 12% (w/v) polyacrylamide SDS gels stained with Coomassie blue or transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA), probed with polyclonal rabbit anti-DnaJ antibody, and developed with goat anti-rabbit IgG/horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (Amersham, Buckinghamshire, UK).

In vivo DnaJ activity assays:
Bacteriophage cI (clear plaque former) and cI dnaJ⁺ transducing plaque-forming assays were performed at 30° as described (KELLEY and GEORGOPOULOS 1997 ). For bacterial viability assays, fresh single-colony plasmid transformants in WKG90 were grown overnight at 30° and serially diluted, and aliquots were spotted on Luria-Bertani (LB) agar plates supplemented with 50 µg/ml ampicillin, with or without L-arabinose. Plates were incubated at 30° and 40° for 20 hr and at 14° for 5 days. Bacterial motility was assessed using fresh single-colony plasmid transformants in MG1655 cbpA dnaJ-Tn10-42 grown to OD₆₀₀ 1.2 in LB broth at 30°. Aliquots (1 µl) were spotted on soft agar plates (1% tryptone, 0.5% NaCl, 0.35% agar), with or without L-arabinose inducer. Motility was analyzed after 6 hr of incubation at 30°. The pBAD22 vector alone and pWKG91 encoding the dnaJ259 (H33Q) inactivating mutation (WALL et al. 1994 ; KELLEY and GEORGOPOULOS 1997 ) were used as negative controls in all assays.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Choice of mutations for structure and function analysis:
The high-resolution structure of the J-domain fold shows that it is stabilized by a core of buried, or partially buried, hydrophobic residues (SZYPERSKI et al. 1994 ; HILL et al. 1995 ; PELLACCHIA et al. 1996 ). Several key core amino acids exhibit multiple long-range interhelical nuclear Overhauser (NOE) connectivities indicating that their alteration could significantly destabilize the protein, leading to unpredictable phenotypes (SZYPERSKI et al. 1994 ). Thus, in our study, we avoided mutations altering any of the amino acids whose side chains comprise the buried core interface of helices I, II, and III. We instead focused our mutational analysis on those solvent-exposed residues that could comprise the J-domain interaction surface with DnaK. Fig 1 depicts schematically the entire collection of amino acid substitutions and deletions analyzed.

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. Summary of the DnaJ J-domain mutants. Other domains of DnaJ are shown as G/F, glycine-phenylalanine rich; Zn, zinc-finger domain; and crosshatching, a less-conserved C-terminal domain. The -helical boundaries are depicted. The mutation type is indicated by rectangles above (point mutations) or below (multiple) the sequence. The hydrophobic core that stabilizes the J-domain structure is composed principally of buried side chains of residues I9, L10, V12, I21, A53, L57 and also Y25, Y54, Y6, Y7, V56, A24, A29, A54 that are peripherally associated with the core.

Double and triple mutants rapidly exclude nonessential regions:
We first constructed a series of adjacent double- or triple-alanine scanning mutants bounded by amino acid residues 2–55 of the minimal J-domain fold to identify rapidly those regions containing important amino acids. The mutations KQD(3-5)AAA, KT(14-15)AA, RE(19-20)AA, MK(30-31)AA, GD(39-40)AA, KE(41-42)AA, KE(48-49)AA, and KE(51-52)AA did not show any discernible effect upon J-domain function in all assays employed (data not shown). Similarly, single-point mutants E8A, S13A, Y32A, E44A, K46A, Y54A, and E55A showed no effect on J-domain function in any of the assays (data not shown).

We observed that mutation EE(17-18)AA measurably affected J-domain function for bacterial growth only at high temperatures, albeit significantly less than the canonical H33Q inactivating mutation, which totally abolishes all J-domain function (WALL et al. 1994 ; data not shown). The EE(17-18)AA mutation conferred normal motility and sensitivity to bacteriophage . When we engineered the separate E17A and E18A point mutations we found that neither single mutation had a discernible phenotype in any assay. E17A and E18A proteins were stable and were produced to levels equivalent to those of wild-type DnaJ (data not shown). In contrast, the EE(17,18)AA double-mutant protein was not detectable either in Coomassie-blue-stained gels or by immunoblot. Most likely, this double amino acid substitution severely disrupts protein stability since residue E17 or E18 acts structurally as the helix II N-cap (PELLACCHIA et al. 1996 ).

Mutations in the flexible helices II-III loop reveal the importance of the ₃₃HPDRN₃₇ pentapeptide loop:
The HPD tripeptide motif, located in the loop joining helices II and III, is highly conserved in all known functional Hsp40 proteins and has been predicted to mediate interaction with DnaK (WILD et al. 1992 ; SZYPERSKI et al. 1994 ). Besides H33Q (dnaJ259) and D35N (dnaJ236), no other mutations with significant phenotypes in the E. coli DnaJ J-domain have been described (WALL et al. 1994 ; SUH et al. 1998 ). We therefore first designed alanine scanning, deletion, and glycine substitution mutants in residues 33–40 surrounding HPD and comprising the loop. Since the J-domain structure shows the helices II and III loop to be highly disordered (PELLACCHIA et al. 1996 ; SUH et al. 1998 ), we chose to alter residues 36–38 to glycine to preserve maximal conformational dihedral space. The results of this study are shown in Table 1.

We found that the deletion of H33, P34, or D35 abolished bacterial growth at high and low temperature, although the steady-state mutant protein levels of all mutants were comparable to those of wild type. Motility and bacteriophage growth were also concomitantly abolished by these mutations (data not shown). Similarly, the P34F point mutation, which is known to inactivate the SV40 J-domain (PEDEN and PIPAS 1992 ), abolished DnaJ function in all assays, despite comparable wild-type steady-state protein levels (Table 1; data not shown). Collectively, these results indicate that each of the contiguous highly conserved HPD residues are essential for function in DnaJ.

The triple glycine substitution mutant, RNQ(36-38)GGG, adjacent to the HPD tripeptide, was defective for J-domain function in all assays, although it showed steady-state protein levels comparable to those of wild-type control. Single glycine substitution of each residue R36, N37, and Q38, within the helices II and III loop, revealed that R36 and N37 were also important for J-domain function, while Q38G behaved phenotypically like wild-type DnaJ. Titration of arabinose inducer levels showed that R36G and N37G could not complement for bacterial growth at 0.01% arabinose, whereas at 0.1% arabinose, both mutants could support bacterial growth at low and high temperatures, although the complementation by R36G was reproducibly less robust than that by N37Q (Table 1). The cluster of contiguous critical residues ₃₃HPDRN₃₇ is noteworthy since mutation of residues 38–40 (QGD) following this pentapeptide had no measurable phenotypic effect (Table 1). We conclude that the ₃₃HPDRN₃₇ pentapeptide constitutes an essential region for J-domain function.

F47, in close proximity to H33, is essential for J-domain function:
The classical mutation H33Q, or the equivalent change in at least eight other Hsp40 proteins, from organisms including yeast and humans, abolishes J-domain biological function (WILD et al. 1992 ; WALL et al. 1994 ; TSAI and DOUGLAS 1996 ; KELLEY and GEORGOPOULOS 1997 ). Since the H33 residue is essential, we next examined closely the phenotypes of substitutions of amino acids whose side chains are in close spatial proximity to H33. The side chains of A29, M30, K31, Y32, E44, and F47 are all in close proximity to H33, on the same face of the J-domain (SZYPERSKI et al. 1994 ). We observed that the point mutation F47A, in an amino acid that is partially exposed and that makes no detectable long-range interhelical NOE contacts with other residues (SZYPERSKI et al. 1994 ), abolished all J-domain activity in every assay (Fig 2). In contrast, mutations A29G, MK(30,31)AA, Y32A, and E44A were phenotypically indistinguishable from wild-type DnaJ in each assay (data not shown). Steady-state protein expression levels of mutants were comparable to those of wild-type DnaJ (Fig 2; data not shown). We conclude from this analysis that F47 in III is an essential residue for J-domain function.

View larger version (62K): In this window In a new window Download PPT slide   Figure 2. Analysis of DnaJ J-domain mutant phenotypes in vivo. (A) Growth temperatures and the fold dilution are indicated. Bacteriophage cI plaque-forming ability is indicated by + if equivalent to wild-type bacteria or - if <10-5 compared to dnaJ+ transducing bacteriophage control. An immunoblot analysis of whole-cell extracts showing the steady-state expression levels of the wild-type and the mutant DnaJ proteins is presented. (B) Representative bacterial motility assay showing the radial growth of MG1655 dnaJ::Tn10 cbpA::kan cells containing pWKG90 (WT) compared to those cells containing vector alone or the DnaJ J-domain mutant (F47A). (C) Ribbon diagram showing the close spatial proximity of the F47 aromatic side chain to H33. The figure was generated using PDB coordinates 1XBL (PELLACCHIA et al. 1996 ) and Swiss PdbViewer (http://www.expasy.ch/sbpdv/) and then was rendered with POV-RAY (http://www.povray.org).

In this respect, it is important to emphasize that we observed no other alteration of residues in III, apart from F47, that resulted in measurable disruption of J-domain function. For example, mutations KE(41,42)AA, E44A, K46A, KE(48,49)AA, KE(51,52)AA, Y54A, and E55A were all phenotypically silent in each assay (data not shown).

Helix II mutations reveal the importance of Y25 and K26:
Helix II is amphipathic with a solvent-exposed face composed of predominantly positively charged side chains, evolutionarily conserved among J-domains (HENNESSY et al. 2000 ). We observed that mutations RE(19,20) AA, L28A, and A29G within the helix II boundaries had no significant effect on J-domain activity in any of our in vivo assays (data not shown). We next examined seven alanine scanning mutants in the positively charged region encompassing residues 21–27, including a quadruple alanine scanning mutant that replaced all four charged side chains. The results of this study are shown in Fig 3.

View larger version (64K): In this window In a new window Download PPT slide   Figure 3. Analysis of J-domain mutant phenotypes in the positively charged region of helix II. (A) The position of mutants is indicated by rectangles as single (above) or multiple mutants (below). (B) Assays were performed as described in the legend to Fig 2A. Motility is indicated by + if cells were comparable to wild-type control and by - if no significant difference from vector control was observed.

We observed that the quadruple mutant RK(22,23)AA/KR(26,27)AA abolished J-domain activity in every assay. Expression of the triple-alanine mutant YKR(25-27)AAA was highly toxic in WGK190 cells and was not investigated further. The double-alanine mutant RK(22,23)AA alone did not show a measurable defect in any assay and could not be distinguished from control cells expressing wild-type DnaJ. In contrast, the double-mutant KR(26,27)AA alone abolished J-domain activity to an extent comparable to the quadruple mutant. These results suggested that the region spanning residues 25–27, but not residues 21–23, was important for J-domain function. To refine this analysis, we next analyzed the phenotypes of the single-alanine mutants Y25A, K26A, and R27A.

As shown in Fig 3, mutant R27A was phenotypically indistinguishable from control wild-type DnaJ, whereas either Y25A or K26A abolished bacterial growth at both low and high temperatures. The phenotype of Y25A was judged more severe since we observed concomitant loss of motility and bacteriophage growth, whereas K26A retained partial activity in both the motility and plaque assays. Importantly, we observed that K26A acted synergistically with the phenotypically silent R27A mutant, resulting in a double mutant with a more severe phenotype than that of either single-mutant clone. This result suggested that the presence of positively charged side chains at position 26–27 was important for DnaJ function. Consistent with this conclusion, a charge reversal mutant, K26E, showed a more severe inactivating phenotype than that of K26A in all assays employed (data not shown). Control immunoblots showed that the steady-state levels of all mutant proteins were equivalent to those of wild-type DnaJ (Fig 3). We conclude from this analysis that the solvent-exposed K26 side chain and the partially exposed Y25 side chain in II are especially important for J-domain function. Taken together, our results reveal a striking demarcation of the contiguous essential loop residues ₃₃HPDRN₃₇, since we observed no measurable phenotypic changes arising with mutants in any of the six residues, ₂₇KLAMKY₃₂, preceding, or the four residues, ₃₈GDKE₄₁, following the ₃₃HPDRN₃₇ pentapeptide.

Mutations of helix IV residues exert no measurable effect on J-domain function:
A fourth helix, helix IV, terminates the J-domain motif, but its structural role in anchoring the essential fold is minimal (SZYPERSKI et al. 1994 ; PELLACCHIA et al. 1996 ). To examine the role of the helices III and IV loop and of the helix IV residues outside the central 2-55 J-domain fold, we engineered five mutations: T58A, TD(58-59)AA, SQ(60-61)AA, KR(62-63)AA, and DQ(66-67)AA (see Fig 4). We observed that all five alanine scanning mutants behaved phenotypically as wild-type DnaJ in each assay. The steady-state levels of the mutant proteins were also comparable to the levels of wild-type DnaJ (data not shown). We also constructed a complete in-frame deletion of residues 58–69 (inclusive) that eliminated the short interhelical loop and all of helix IV from the dnaJ coding sequence. The resulting mutant, DnaJ (58-69), also showed no significant phenotypic difference from wild-type DnaJ in any of our in vivo assays under our standard laboratory conditions (Fig 4).

View larger version (56K): In this window In a new window Download PPT slide   Figure 4. Analysis of J-domain mutant phenotypes in helices III and IV loop and in helix IV. (A) The position of mutants is indicated by rectangles as single (above) or multiple mutants (below). (B) The functional complementation assays were performed as described in the legend to Fig 2A.

Hdj1 inactivating mutations at the corresponding position of critical DnaJ residues:
The overall J-domain fold of the human Hdj1 (QIAN et al. 1996 ) is highly similar to the E. coli DnaJ J-domain (PELLACCHIA et al. 1996 ) and the two proteins share 54% amino acid identity in the J-domain (Fig 5D). We constructed a chimeric DnaJ expression plasmid, pGP18, which substituted the human Hdj1 J-domain coding sequence in place of the native E. coli DnaJ J-domain sequence. The hybrid DnaJ could fully subsititute for native DnaJ in each in vivo assay, indicating that the human J-domain could functionally substitute for the bacterial J-domain (in Fig 6, compare DnaJ and Hdj1). Next, we asked whether mutating any of the Hdj1 residues at the corresponding positions of the important DnaJ residues uncovered in our screen would result in similar inactivating phenotypes. To this end, alanine and glycine substitution mutants were engineered for Hdj1 Y24A, RR(25,26)AA, K35G, N36G, and F45A and tested (see alignment, Fig 5D). The numbering for the hdj1 mutants uses the methionine start codon as 0, consistent with the PDB accession 1HDJ (QIAN et al. 1996 ). The results are shown in Fig 6. We observed that Y24A, RR(25,26)AA, and F45A abolished J-domain function for growth at both high and low temperatures. At higher arabinose concentrations, slow-growing colonies were observed for Y24A and a few colonies at 10^-3 dilution were observed for F45A. The mutant Y24A weakly reduced plaque-forming efficiency, whereas RR(25,26)AA and F45A completely abolished plaque formation (efficiency of plating <10^-5) under all conditions tested. The Hdj1 mutant K35G behaved like its DnaJ counterpart, R36G, by showing marked effects on growth and plaque formation. In contrast and unlike the DnaJ N37G mutant, the Hdj1 N36G behaved like wild type in all assays (Fig 6).

View larger version (19K): In this window In a new window Download PPT slide   Figure 5. Ribbon projection of E. coli DnaJ and human Hdj1 J-domains showing the tight surface clustering of important residues identified by mutational analysis. (A and B) Two views of the DnaJ J-domain together with marked essential side chain residues. B is a top projection down the z-axis of A, following a 90° clockwise rotation around the z-axis. (C) Human Hdj1 J-domain showing the location of essential residues in the same perspective as A. Figures were generated as described in the legend to Fig 2B. (D) Alignment of the E. coli J-domain and the human Hdj1 J-domain sequences. Identical residues are black, and conserved substitutions are gray. The location of the mutations is shown by arrows. Black indicates a change for alanine, and gray indicates a change for glycine.

View larger version (67K): In this window In a new window Download PPT slide   Figure 6. Analysis of J-domain mutant phenotypes in the Hdj1 J-domain. The functional complementation assays were performed as described in the legend to Fig 2A.

Mapping essential residues on the J-domain structure:
We mapped the critical residues Y25, K26, ₃₃HPDRN₃₇, and F47 that we identified on the three-dimensional structure of the E. coli J-domain (Fig 5A and Fig B). Interestingly, these residues cluster in a small region on the same face of the J-domain. We also mapped the critical residues Y24, RR(25,26), K35, and F45 of Hdj1 on its J-domain three-dimensional structure (Fig 5C). As with DnaJ, these residues in Hdj1 also cluster together on the same spatial solvent-exposed face of the J-domain. Since most of other DnaJ mutants tested resulted in no detectable phenotype under any of our in vivo experimental conditions, we conclude that this compact region must encompass the totality, or major functional portion, of the J-domain interacting surface with DnaK.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study, we have used an exhaustive directed mutagenesis to identify residues in the E. coli DnaJ J-domain that are required for its cochaperone function with DnaK in vivo. Our extensive mutagenic screen has uncovered a new set of residues (Y25, K26, P34, R36, N37, and F47), in addition to those previously described, namely, dnaJ259 (H33Q) and dnaJ236 (D35N), that are important for J-domain function. Interestingly, all of these critical residues, located in helix II, helix III, and the helices II and III loop, project their side chains on the same solvent-exposed face of the J-domain. These same amino acids are highly conserved in the human Hdj1 J-domain and occupy a similar three-dimensional spatial arrangement (QIAN et al. 1996 ). Mutation of Hdj1 J-domain residues, in the context of a chimeric DnaJ-Hdj1 molecule, closely mirrors the corresponding mutation of DnaJ J-domain residues. This finding underscores the notion that the principal contact surface between a J-domain and the Hsp70 ATPase domain has been preserved through evolution and suggests that predictable inactivating mutations can be introduced in other J-domain proteins. We propose, therefore, that this region represents the principal contact surface interface between the J-domain and the Hsp70 ATPase domain.

It is surprising that we identified so few essential amino acid residues. Of the 48 residues mutated in the J-domain, only 8 resulted in a significant phenotype in our in vivo assays. These results, together with the fact that the E. coli DnaJ J-domain can be functionally replaced with a variety of distantly related J-domains—for instance, mammalian papovavirus T antigens (KELLEY and GEORGOPOULOS 1997 ; BERJANSKII et al. 2000 ), Mdj1 (DELOCHE et al. 1997 ), DjlA (GENEVAUX et al. 2001 ), Ydj1, and the bovine or Torpedo cysteine string protein (W. L. KELLEY, unpublished data)—would explain the apparent substitution plasticity observed with extensive multiple J-domain alignments (HENNESSY et al. 2000 ). Unlike E. coli DnaJ, SV40 T antigen tolerates only limited J-domain exchange from a related human virus JC, but does not function with J-domains from either E. coli DnaJ or Ydj1 (SULLIVAN et al. 2000 ).

Our in vivo genetic data are consistent with in vitro biochemical and biophysical studies that have attempted to map the J-domain interaction surface. Peptide competition studies using native Saccharomyces cerevisiae Ydj1p (Hsp40)-Ssa1p (Hsp70) interaction also predicted that the hinge region between II and III, including HPD, mediated productive interaction with Hsp70 (CYR et al. 1992 ; TSAI and DOUGLAS 1996 ). Analysis of exclusively backbone amide proton chemical shifts by NMR perturbation mapping using E. coli DnaJ 2-75-DnaK titration led GREENE et al. 1998 to suggest that the minimal J-domain interaction surface could be composed of residues 2–35, including the HPD tripeptide. Out of the 17 residues identified by GREENE et al. 1998 that showed significant amide perturbation upon titration with DnaK(MgATP), 14 were mutated in our analysis, and only 4 (Y25, K26, H33, and D35) resulted in loss of function in vivo. Polypeptide backbone amide interactions, if important in vivo, may have been preserved in our mutants.

An electrostatic surface plot predicted that the DnaJ J-domain was highly asymmetric with respect to charge and that the positive face of helix II could provide an important recognition surface for the predominantly negative potential of the DnaK ATPase domain (PELLACCHIA et al. 1996 ; GREENE et al. 1998 ). Consistent with this prediction, ZIEGELHOFFER et al. 1995 noted that stimulation of the S. cerevisiae Ssa1p (Hsp70) ATPase by Ydj1p (Hsp40) was significantly affected by KCl titration (ZIEGELHOFFER et al. 1995 ). Our data support the prediction that the positive face of helix II is important for J-domain function and narrow it to residue K26 as the only critical one. This finding is also in complete agreement with the prediction of BERJANSKII et al. 2000 that the b2 position of the helices II and III coiled coil of polyomavirus T antigen J-domain is the most essential positive charge position in helix II governing J-domain interactions with Hsp70 (BERJANSKII et al. 2000 ). Indeed, polyomavirus T antigen J-domain can functionally substitute for DnaJ J-domain in our in vivo growth assays, despite lacking these conserved positive charge residues, except K35, which is equivalent to DnaJ K26 (BERJANSKII et al. 2000 ).

The mechanistic role of the F47 residue is presently unclear. One possibility is that a bulky side chain projecting at this position may sterically constrain the limits of the helices II and III loop movement. This phenylalanine is highly conserved and positive charge residues flanking this residue have been proposed to play a role in mediating interaction with Hsp70 (HENNESSY et al. 2000 ). In our study, mutation of the lysines flanking F47, and indeed nearly all surface-exposed residues of helix III, had no detectable effect in vivo. In contrast, in an attempt to understand Hsp40-Hsp70 partner specificity, SCHLENSTEDT et al. 1995 showed that a single amino acid change, K42V, which is the positionally equivalent adjacent residue to DnaJ F47, could partially restore activity to an S. cerevisiae Sec63 chimera harboring a nonfunctional endoplasmic reticulum lumenal J-domain derived from cytoplasmic Sis1 (SCHLENSTEDT et al. 1995 ).

Two in vitro studies suggest that the conserved ₆₁QKRAA₆₅ motif of helix IV might contribute to binding to DnaK, although likely in the substrate cavity (AUGER et al. 1996 ; SUH et al. 1998 ). Our work shows that single and double mutations along this region, as well as deletion of the entire IV, had no significant phenotype. Although we cannot exclude that helix IV plays an as yet undetermined role in the biology of DnaJ, these results indicate that amino acid residues of IV are not required for DnaJ to perform as DnaK cochaperone in vivo. Although polyomavirus and SV40 J-domains can replace DnaJ J-domain in vivo (KELLEY and GEORGOPOULOS 1997 ; BERJANSKII et al. 2000 ), sequence comparison with the J-domain of either DnaJ or Hdj1 shows no significant conservation in the region of helix IV. In addition, the three-dimensional structures of the J-domains of polyomavirus and SV40 T antigens reveal a longer helix III and a long loop in place of helix IV, when compared to those of DnaJ and Hdj1. These results are in complete agreement with our findings.

Finally, the E. coli J-domain cannot by itself stimulate DnaK's ATPase and requires either an adjacent contiguous sequence, such as the Gly-Phe domain (G/F; WALL et al. 1994 ), or a costimulatory peptide/substrate that can interact with the DnaK substrate binding domain (SZABO et al. 1994 ; KARZAI and MCMACKEN 1996 ; SUH et al. 1998 ). A truncated DnaJ, containing residues 1–108 (DnaJ12), can perform some of the functions of wild-type DnaJ in vivo and can stimulate DnaK ATPase in vitro, suggesting that it contains minimal determinants for cochaperone function (WALL et al. 1994 ; LIBEREK et al. 1995 ). Using NMR methods, HUANG et al. 1999 compared the structural changes in the J-domain that occur in the presence (DnaJ2-104) or absence (DnaJ2-78) of the adjacent G/F domain. Their results led to the proposal that the helices II and III loop and portions of helix II could adapt alternative conformations in the presence of G/F. In particular, a plot of the dihedral angles of residues in the helices II and III loop region revealed the possibility of a concerted hinge movement. Strikingly, the hinge region identified by HUANG et al. 1999 , ₃₂YHPDR₃₆, and the contiguous ₃₃HPDRN₃₇ pentapeptide harboring essential residues revealed in our study are nearly identical. The intriguing possibility is that this flexible loop/hinge region assumes alternative conformations that mediate reversible contact when paired with DnaK.

	FOOTNOTES

² Present address: Division des Maladies Infectieuses, Hôpital Cantonal de Genève, CH-1211 Geneva 14, Switzerland.

	ACKNOWLEDGMENTS

We thank Drs. Sam Landry and Maurizio Pellecchia for helpful discussions and Kyle Tanner for advice with PovRay. This work was supported by grant FN-31-065403.01 from the Swiss National Science Foundation and the Canton of Geneva.

Manuscript received June 16, 2002; Accepted for publication August 9, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AUGER, I., J. M. ESCOLA, J. P. GORVEL, and J. ROUDIER, 1996  HLA-DR4 and HLA-DR10 motifs that carry susceptibility to rheumatoid arthritis bind 70-kD heat shock proteins. Nat. Med. 2:306-310.[Medline]

BERJANSKII, M. V., M. I. RILEY, A. XIE, V. SEMENCHENKO, and W. R. FOLK et al., 2000  NMR structure of the N-terminal J domain of murine polyomavirus T antigens: implications for DnaJ-like domains and for mutations of T antigens. J. Biol. Chem. 275:36094-36103.[Abstract/Free Full Text]

BUKAU, B. and A. L. HORWICH, 1998  The Hsp70 and Hsp60 chaperone machines. Cell 92:351-366.[Medline]

CYR, D. M., X. LU, and M. G. DOUGLAS, 1992  Regulation of eukaryotic hsp70 function by a dnaJ homolog. J. Biol. Chem. 267:20927-20931.[Abstract/Free Full Text]

DELOCHE, O., W. L. KELLEY, and C. GEORGOPOULOS, 1997  Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli.. J. Bacteriol. 179:6066-6075.[Abstract/Free Full Text]

GASSLER, C. S., A. BUCHBERGER, T. LAUFEN, M. P. MAYER, and H. SCHRODER et al., 1998  Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone Proc. Natl. Acad. Sci. USA 95:15229-15234.[Abstract/Free Full Text]

GASSLER, C. S., T. WIEDERKEHR, D. BREHMER, B. BUKAU, and M. P. MAYER, 2001  Bag-1M accelerates nucleotide release for human Hsc70 and Hsp70 and can act concentration-dependent as positive and negative cofactor. J. Biol. Chem. 276:32538-32544.[Abstract/Free Full Text]

GENEVAUX, P., A. WAWRZYNOW, M. ZYLICZ, C. GEORGOPOULOS, and W. L. KELLEY, 2001  DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction. J. Biol. Chem. 276:7906-7912.[Abstract/Free Full Text]

GREENE, M. K., K. MASKOS, and S. J. LANDRY, 1998  Role of the J-domain in the cooperation of Hsp40 with Hsp70. Proc. Natl. Acad. Sci. USA 95:6108-6113.[Abstract/Free Full Text]

HARTL, F. U., 1996  Molecular chaperones in cellular protein folding. Nature 381:571-580.[Medline]

HENNESSY, F., M. E. CHEETHAM, H. W. DIRR, and G. L. BLATCH, 2000  Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins. Cell Stress Chaperones 5:347-358.[Medline]

HIGUCHI, R., B. KRUMMEL, and R. K. SAIKI, 1988  A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16:7351-7367.[Abstract/Free Full Text]

HILL, R. B., J. M. FLANAGAN, and J. H. PRESTEGARD, 1995  ¹H and ¹⁵N magnetic resonance assignments, secondary structure, and tertiary fold of Escherichia coli DnaJ (1-78). Biochemistry 34:5587-5596.[Medline]

HUANG, K., R. GHOSE, J. M. FLANAGAN, and J. H. PRESTEGARD, 1999  Backbone dynamics of the N-terminal domain in E. coli DnaJ determined by ¹⁵N- and ¹³CO-relaxation measurements. Biochemistry 38:10567-10577.[Medline]

KARZAI, A. W. and R. MCMACKEN, 1996  A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. J. Biol. Chem. 271:11236-11246.[Abstract/Free Full Text]

KELLEY, W. L., 1998  The J-domain family and the recruitment of chaperone power. Trends Biochem. Sci. 23:222-227.[Medline]

KELLEY, W. L. and C. GEORGOPOULOS, 1997  The T/t common exon of simian virus 40, JC, and BK polyomavirus T antigens can functionally replace the J-domain of the Escherichia coli DnaJ molecular chaperone. Proc. Natl. Acad. Sci. USA 94:3679-3684.[Abstract/Free Full Text]

KIM, H.-Y., B.-Y. AHN, and Y. CHO, 2001  Structural basis for the inactivation of retinoblastoma tumor suppressor by SV40 large T antigen. EMBO J. 20:295-304.[Medline]

KUNKEL, T. A., J. D. ROBERTS, and R. A. ZAKOUR, 1987  Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367-383.[Medline]

LIBEREK, K., J. MARSZALEK, D. ANG, C. GEORGOPOULOS, and M. ZYLICZ, 1991  Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc. Natl. Acad. Sci. USA 88:2874-2878.[Abstract/Free Full Text]

LIBEREK, K., D. WALL, and C. GEORGOPOULOS, 1995  The DnaJ chaperone catalytically activates the DnaK chaperone to specifically bind the ³² heat shock transcriptional regulator. Proc. Natl. Acad. Sci. USA 92:6224-6228.[Abstract/Free Full Text]

LYMAN, S. K. and R. SCHEKMAN, 1997  Binding of secretory precursor polypeptides to a translocon subcomplex is regulated by BiP. Cell 88:85-96.[Medline]

MISSELWITZ, B., O. STAECK, and T. A. RAPOPORT, 1998  J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences. Mol. Cell 2:593-603.[Medline]

PEDEN, K. W. and J. M. PIPAS, 1992  Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen. Virus Genes 6:107-118.[Medline]

PELLACCHIA, M., T. SZYPERSKI, D. WALL, C. GEORGOPOULOS, and K. WÜTHRICH, 1996  NMR structure of the J-domain and the Gly/Phe-rich region of the Escherichia coli DnaJ chaperone. J. Mol. Biol. 260:236-250.[Medline]

QIAN, Y. Q., D. PATEL, F.-U. HARTL, and D. J. MCCOLL, 1996  Nuclear magnetic resonance solution structure of the human Hsp40 (HDJ-1) J-domain. J. Mol. Biol. 260:224-235.[Medline]

RAABE, T. and J. L. MANLEY, 1991  A human homologue of the Escherichia coli DnaJ heat shock protein. Nucleic Acids Res. 19:6645.[Free Full Text]

SCHLENSTEDT, G., S. HARRIS, B. RISSE, R. LILL, and P. A. SILVER, 1995  A yeast DnaJ homologue, Scj1p, can function in the endoplasmic reticulum with BiP/Kar2p via a conserved domain that specifies interactions with Hsp70's. J. Cell Biol. 129:979-988.[Abstract/Free Full Text]

SHI, W., Y. ZHOU, J. WILD, J. ADLER, and C. A. GROSS, 1992  DnaK, DnaJ, and GrpE are required for flagellum synthesis in Escherichia coli.. J. Bacteriol. 174:6256-6263.[Abstract/Free Full Text]

SUH, W. C., W. F. BURKHOLDER, C. Z. LU, X. ZHAO, and M. E. GOTTESMAN et al., 1998  Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ. Proc. Natl. Acad. Sci. USA 95:15223-15228.[Abstract/Free Full Text]

SULLIVAN, C. S., J. D. TREMBLAY, S. W. FEWELL, J. A. LEWIS, and J. L. BRODSKY et al., 2000  Species-specific elements in the large T-antigen J domain are required for cellular transformation and DNA replication by simian virus 40. Mol. Cell. Biol. 20:5749-5757.[Abstract/Free Full Text]

SZABO, A., T. LANGER, H. SCHRÖDER, J. FLANAGAN, and B. BUKAU et al., 1994  The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system-DnaK, DnaJ, and GrpE. Proc. Natl. Acad. Sci. USA 91:10345-10349.[Abstract/Free Full Text]

SZYPERSKI, T., M. PELLACCHIA, D. WALL, C. GEORGOPOULOS, and K. WÜTHRICH, 1994  NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2–108), containing the highly conserved J-domain. Proc. Natl. Acad. Sci. USA 91:11343-11347.[Abstract/Free Full Text]

TSAI, J. and M. G. DOUGLAS, 1996  A conserved HPD sequence of the J-domain is necessary for YDJ1 stimulation of hsp70 ATPase activity at a site distinct from substrate binding. J. Biol. Chem. 271:9347-9354.[Abstract/Free Full Text]

UEGUCHI, C., T. SHIOZAWA, M. KAKEDA, H. YAMADA, and T. MIZUNO, 1995  A study of the double mutation of dnaJ and cbpA, whose gene products function as molecular chaperones in Escherichia coli.. J. Bacteriol. 177:3894-3896.[Abstract/Free Full Text]

WALL, D., M. ZYLICZ, and C. GEORGOPOULOS, 1994  The NH₂-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for replication. J. Biol. Chem. 269:5446-5451.[Abstract/Free Full Text]

WILD, J., E. ALTMAN, T. YURA, and C. A. GROSS, 1992  DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli.. Genes Dev. 6:1165-1172.[Abstract/Free Full Text]

ZIEGELHOFFER, T., P. LOPEZ-BUESA, and E. A. CRAIG, 1995  The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide substrates. J. Biol. Chem. 270:10412-10419.[Abstract/Free Full Text]

This article has been cited by other articles:

				C. Yang, H. A. Owen, and P. Yang Dimeric heat shock protein 40 binds radial spokes for generating coupled power strokes and recovery strokes of 9 + 2 flagella J. Cell Biol., January 28, 2008; 180(2): 403 - 415. [Abstract] [Full Text] [PDF]

				C. M. Wright, S. W. Fewell, M. L. Sullivan, J. M. Pipas, S. C. Watkins, and J. L. Brodsky The Hsp40 Molecular Chaperone Ydj1p, Along With the Protein Kinase C Pathway, Affects Cell-Wall Integrity in the Yeast Saccharomyces cerevisiae Genetics, April 1, 2007; 175(4): 1649 - 1664. [Abstract] [Full Text] [PDF]

				R. S. Ullers, D. Ang, F. Schwager, C. Georgopoulos, and P. Genevaux Trigger Factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli PNAS, February 27, 2007; 104(9): 3101 - 3106. [Abstract] [Full Text] [PDF]

				G. C. Cajo, B. E. Horne, W. L. Kelley, F. Schwager, C. Georgopoulos, and P. Genevaux The Role of the DIF Motif of the DnaJ (Hsp40) Co-chaperone in the Regulation of the DnaK (Hsp70) Chaperone Cycle J. Biol. Chem., May 5, 2006; 281(18): 12436 - 12444. [Abstract] [Full Text] [PDF]

				K. A. Whalen, R. de Jesus, J. A. Kean, and B. S. Schaffhausen Genetic Analysis of the Polyomavirus DnaJ Domain J. Virol., August 1, 2005; 79(15): 9982 - 9990. [Abstract] [Full Text] [PDF]

				F. Hennessy, W. S. Nicoll, R. Zimmermann, M. E. Cheetham, and G. L. Blatch Not all J domains are created equal: Implications for the specificity of Hsp40-Hsp70 interactions Protein Sci., July 1, 2005; 14(7): 1697 - 1709. [Abstract] [Full Text] [PDF]

				J. M. Gruschus, L. E. Greene, E. Eisenberg, and J. A. Ferretti Experimentally biased model structure of the Hsc70/auxilin complex: Substrate transfer and interdomain structural change Protein Sci., August 1, 2004; 13(8): 2029 - 2044. [Abstract] [Full Text] [PDF]

				L. Rohmer, D. S. Guttman, and J. L. Dangl Diverse Evolutionary Mechanisms Shape the Type III Effector Virulence Factor Repertoire in the Plant Pathogen Pseudomonas syringae Genetics, July 1, 2004; 167(3): 1341 - 1360. [Abstract] [Full Text] [PDF]

				P. D. D'Silva, B. Schilke, W. Walter, A. Andrew, and E. A. Craig J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix PNAS, November 25, 2003; 100(24): 13839 - 13844. [Abstract] [Full Text] [PDF]

				K. Linke, T. Wolfram, J. Bussemer, and U. Jakob The Roles of the Two Zinc Binding Sites in DnaJ J. Biol. Chem., November 7, 2003; 278(45): 44457 - 44466. [Abstract] [Full Text] [PDF]

				P. Genevaux, F. Lang, F. Schwager, J. V. Vartikar, K. Rundell, J. M. Pipas, C. Georgopoulos, and W. L. Kelley Simian Virus 40 T Antigens and J Domains: Analysis of Hsp40 Cochaperone Functions in Escherichia coli J. Virol., October 1, 2003; 77(19): 10706 - 10713. [Abstract] [Full Text] [PDF]