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. 2001 Mar 1;20(5):1042-50.
doi: 10.1093/emboj/20.5.1042.

Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone

S Rüdiger  1 , J Schneider-Mergener, B Bukau
Affiliations

Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone

S Rüdiger et al. EMBO J. .

Abstract

The evolutionarily conserved DnaJ proteins are essential components of Hsp70 chaperone systems. The DnaJ homologue of Escherichia coli associates with chaperone substrates and mediates their ATP hydrolysis-dependent locking into the binding cavity of its Hsp70 partner, DnaK. To determine the substrate specificity of DnaJ proteins, we screened 1633 peptides derived from 14 protein sequences for binding to E.coli DnaJ. The binding motif of DnaJ consists of a hydrophobic core of approximately eight residues enriched for aromatic and large aliphatic hydrophobic residues and arginine. The hydrophobicity of this motif explains why DnaJ itself can prevent protein aggregation. Although this motif shows differences from DnaK's binding motif, DnaJ and DnaK share the majority of binding peptides. In contrast to DnaK, DnaJ binds peptides consisting of L- and D-amino acids, and therefore is not restricted by backbone contacts. These features allow DnaJ to scan hydrophobic protein surfaces and initiate the functional cycle of the DnaK system by associating with hydrophobic exposed patches and subsequent targeting of DnaK to these or to hydrophobic patches in spatial neighbourhood.

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Figures

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Fig. 1. DnaJ binding to cellulose-bound peptide scans. Peptide scans derived from sequences of DnaA, λP, p53 and luciferase were screened for DnaJ binding. Last spots of rows (right) and NH2-terminal residues of peptides of the first spots of rows (left) are indicated.
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Fig. 2. Amino acid distribution in peptide-scanning libraries. For 1633 peptides representing 14 protein sequences the relative amino acid occurrence was determined. (A) Normalized affinity of DnaJ for the peptides investigated. Peptides are ordered according to their affinity for DnaJ. For the statistical analysis, two sets of peptides of high affinity for DnaJ (relative affinity >40; black bars) and low affinity for DnaJ (relative affinity <10; light bars) were selected. (B) Peptides with high affinity for DnaJ (dark bars) compared with DnaK-binding peptides identified in a previous study (Rüdiger et al., 1997b; light bars). The number for each amino acid is normalized to its occurrence in the whole peptide library (=100). (C) Sequence alignment of DnaJ-binding regions. Sixty-two DnaJ-binding regions each constituting a single strong DnaJ-binding site were aligned. Hydrophobic cores were anchored with a large hydrophobic or aromatic residue at position 10 by shifting the sequences by up to two residues. The frequency of acidic (white bars), large hydrophobic and aromatic (black bars) and basic residues (grey bars) at each position is given as a percentage. Large hydrophobic and aromatic residues are enriched between positions 10 and 17.
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Fig. 3. Comparison of binding of DnaK and DnaJ to peptides. (A) DnaJ- and DnaK-binding peptides in luciferase are in most cases identical. Luciferase-derived peptides that were screened for DnaJ binding (Figure 1) are represented as bars. The length of the bars corresponds to the affinity of the peptide for DnaJ. Black bars represent peptides that were classified as DnaK binders in a previous study (Rüdiger et al., 1997b). (B) Peptides that bind to DnaJ but not to DnaK contain several large hydrophobic and aromatic residues (Leu, Ile, Phe, Trp and Tyr; black boxes) with acidic residues (Glu and Asp; white circles) in between. Basic residues (Arg and Lys) are represented as grey circles. (C) DnaJ can compete with DnaK–peptide binding in solution. A complex of DnaK (0.1 µM) and the peptide σ32-Q132-Q144-C-IAANS (0.5 µM) was titrated with DnaJ (concentration as indicated). The fluorescence of the DnaK–peptide interaction was obtained by subtracting the fluorescence of the DnaJ–peptide interaction in the absence of DnaK from the total signal.
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Fig. 4. Recognition of the peptide backbone by DnaK but not DnaJ. (A) Illustration of the stereochemical differences between peptides of the same sequence but authentic or inversed sequence direction, or composed of l- or d-peptides. The hexagon and the wavy line symbolize the coupling of the C-terminus of each peptide via a linker to cellulose. The light grey forms symbolize the stereochemical connection of three different side chains of hypothetical tripeptides. The backbone atoms are indicated, except carbon. (B) Peptide scans derived from the sequences of λ CI were screened for binding to DnaJ (a–d) and DnaK (e–h). The peptides were synthesized using l- (a, c, e and g) or d-amino acids (b, d, f and h) and with authentic (e.g. H2N-STKKKPLTQEQLE-COO-cellulose; a, b, e and f) or inverse sequence (e.g. H2N-ELQEQTLPKKKTS-COO-cellulose; c, d, g and h).
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Fig. 5. Common and divergent features of substrate recognition of DnaJ and DnaK. (A) The binding motifs of DnaK and DnaJ in substrates. Positions with contributions of large hydrophobic or aromatic residues (Hy) or positively charged residues (+; the size of the area is proportional to the weight of these contributions) are indicated. While DnaK preferentially binds hydrophobic cores of four or five residues in length and positively charged residues in the flanking regions increase binding (Rüdiger et al., 1997b), DnaJ binds to longer hydrophobic segments (approximately eight residues in length). (B) Segments with denatured polypeptide sequences that bind to both DnaJ (J) and DnaK (K) are characterized by longer stretches enriched in large hydrophobic and aromatic residues (grey tubes) and basic residues (+) neighbouring such stretches, while short hydrophobic stretches bind only to DnaK. Longer stretches with acidic residues in between bind only to DnaJ. (C) Two modes of DnaJ interaction with substrates allow targeting of DnaK to unfolded proteins. Mode 1: DnaJ (J) binds to a hydrophobic segment (dark grey stretch) on the surface of a chaperone substrate that is subsequently bound by DnaK (K) (‘handover’). DnaJ dissociates after stimulation of DnaK’s ATPase; the spike indicates this functional interaction. Mode 2: DnaJ (J) binds to hydrophobic segments (dark grey stretch) on the surface of a chaperone substrate that are spatially near the stretch that is subsequently bound by DnaK (K). DnaJ dissociates after stimulation of DnaK’s ATPase; the spike indicates this functional interaction.
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Fig. 6. Structures of the substrate-binding domains. (A) Domain structures of DnaJ from E.coli and Sis1 from S.cerevisiae. J, J domain; blank segments, G/F motif; grey segments, linker (DnaJ) or G/M motif (Sis1); Zn, zinc-finger domain; C, C-terminal domain. (B) Structure of the substrate-binding domain of Sis1 (C-terminal domain; Sha et al., 2000). (C and D) Structure of the substrate-binding domains of DnaJ. (C) Model of the C-terminal domain. (D) Zinc-finger domain (Martinez-Yamout et al., 2000). Hydrophobic side chains (Ile, Leu, Val, Phe, Trp, Tyr, Ala and Met) are coloured yellow in the space-filling representations (done by InsightII). Red circles indicate the sites we propose as most likely to be responsible for substrate binding according to our biochemical data. The electrostatic potential was rendered on the surface of each domain using Grasp (Nicholls et al., 1993). Red and blue indicate acidic and basic surface regions, respectively. In (C) the green quadrangles indicate the parts of the C-terminal domain of DnaJ in which residues corresponding to the N-terminal residues of the C-terminal domain of Sis1 are missing.
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References

    1. Banecki B., Liberek,K., Wall,D., Wawrzynów,A., Georgopoulos,C., Bertoli,E., Tanfani,F. and Zylicz,M. (1996) Structure–function analysis of the zinc finger region of the DnaJ molecular chaperone. J. Biol. Chem., 271, 14840–14848. - PubMed
    1. Ellis R.J. and Hartl,F.U. (1999) Principles of protein folding in the cellular environment. Curr. Opin. Struct. Biol., 9, 102–110. - PubMed
    1. Feifel B., Schönfeld,H.-J. and Christen,P. (1998) d-peptide ligands for the co-chaperone DnaJ. J. Biol. Chem., 273, 11999–12002. - PubMed
    1. Frank R. (1992) Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron, 48, 9217–9232.
    1. Gamer J., Multhaup,G., Tomoyasu,T., McCarty,J.S., Rüdiger,S., Schönfeld,H.-J., Schirra,C., Bujard,H. and Bukau,B. (1996) A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the E.coli heat shock transcription factor σ32. EMBO J., 15, 607–617. - PMC - PubMed

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