Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

Abstract

Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I-IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.

Fig. 1

Plasmid pRJ-B used for deriving the constructs encoding Agt DnaJ chimeric proteins. (A) A plasmid map of pRJ-B: a BstBI restriction site was engineered into the pRJ30 plasmid between the coding regions for the Agt DnaJ J-domain and GF region by silent mutation of the codon for Phe74 to produce the pRJ-B plasmid. The positions of the BamHI and BstBI restriction endonuclease sites used for swapping the J-domain coding regions in the creation of the Agt DnaJ chimeric proteins are indicated. (B) Codon optimised Pfj1 J-domain coding sequence. The translated J-domain protein sequence is highlighted and the BamHI and BstBI restriction endonuclease sites used for insertion of the coding region for the J-domain into the pRJ-B plasmid are underlined.

Fig. 2

J-domains and conserved J-domain residues targeted for analysis. (A) Structure aided alignment of J-domains analyzed in this study. Predicted α-helices are underlined. Highly conserved charged residues are indicated by arrows. EC—E. coli DnaJ [GenBank accession number: P31689] residues 2–70; AT—A. tumefaciens DnaJ [AAR84666.1] residues 2–70; TC—T. cruzi Tcj3 [AAC18896] residues 2–71; P1—P. falciparum Pfj1 [NP_702750.1] residues 57–125; P4—P. falciparum Pfj4 [BAB17689] residues 2–71; HS—Homo sapiens HSJ1 [NP_001034639] residues 2–69; E1—Mus musculus ERj1 [NP_031895] residues 57–125; E3—H. sapiens ERj3 [NP_057390] residues 21–90; E5—H. sapiens ERj5 [NP_061854] residues 31–99; Con—consensus sequence of the aligned J-domain residues. Con#—consensus numbering used for all sequences in this paper, equivalent to the corresponding E. coli DnaJ residue numbering. *—Proteins of known structure. The numbers on the left-hand side of the alignment represent the positions of the first amino acid in each sequence. Sequences were aligned using ClustalW (Thompson, Higgins, & Gibson, 1994) and then adjusted by hand using available structural data. (B) The ClustalW alignment is represented as an unrooted radial tree using TreeView (Page, 1996). The protein names are indicated at the ends of the branches, and apart from E. coli DnaJ (EcDnaJ), the protein names are indicated by the standard abbreviations. (C) Locations of amino acid residues targeted for substitution shown on a ribbon representation of the known tertiary structure of E. coli DnaJ J-domain (PDB:1XBL). The structure is shown from two different orientations. Helices I–IV are indicated and the highlighted residues are shown as sticks and labelled using the single letter code. The ribbon representations of the structures were rendered using PyMol version 0.98 (DeLano, 2005).

Fig. 3

All the J-domain chimeras of Agt DnaJ, apart from ERj1-J-Agt-DnaJ, were able to reverse the thermosensitivity of E. coli OD259. Plasmids encoding each of the chimeric Hsp40s were transformed into the temperature sensitive strain E. coli OD259. Cells were diluted sequentially, spotted onto agar plates supplemented with IPTG and grown at the non-stress temperature of 30 °C and stress temperature of 40 °C. The dilution factor is indicated above each growth panel. The ability of each protein to compensate for the lack of DnaJ and CbpA was investigated by monitoring the reversal of thermosensitivity under stress temperature conditions at 40 °C. The proteins produced in the cells are indicated with an abbreviation on the left-hand side of each growth panel: +ve—Agt DnaJ; −ve—Agt DnaJ-H33Q; Tcj3—Tcj3-J-Agt-DnaJ; Pfj1—Pfj1-J-Agt-DnaJ; Pfj4—Pfj4-J-Agt-DnaJ; HSJ1—HSJ1-J-Agt-DnaJ; ERj1—ERj1-J-Agt-DnaJ; ERj3—ERj3-J-Agt-DnaJ; ERj5—ERj5-J-Agt-DnaJ. The levels of chimeric protein production in E. coli OD259 were determined by Western analysis (W).

Fig. 4

The R26A helix II mutation of the ERj1 J-domain disrupts binding to BiP, but not as extensively as the H33Q mutation of the HPD motif. (A) SDS-PAGE analysis of pull down assays conducted to determine the relative binding to BiP (0.5 μM) of equimolar concentrations of immobilized GST-ERj1-J (J), GST-ERj1-J-R26A (J-R26A) and GST-ERj1-J-H33Q (J-H33Q) in the presence (+) and absence (−) of ATP (2 mM). The input BiP is indicated (i) and the positions of co-purifying BiP and GST-ERj1-J and its derivatives are indicated by arrows. (B) Graphical presentation of the SPR data for ERj1-J (solid circles) and ERj1-J-R26A (open circles) binding to BiP. Four hundred response units of GST, GST-ERj1-J and GST-ERj1-J-R26A were bound to anti-GST antibodies covalently attached to a CM5 sensor chip. The GST fusion proteins were separately bound to the antibodies in the measuring cell, while the GST was bound to the antibodies in the reference cell. A number of different BiP solutions covering a range of concentrations (hamster BiP; 0.25, 0.75, 1.0 and 2.0 μM) were passed over the sensor chip in the presence of ATP. Each BiP application was followed by the application of running buffer containing ATP, thus allowing the association and dissociation kinetics to be followed. The maximum response units recorded for the association phase were plotted against concentration, and a curve fitted to the data by non-linear regression assuming one-site binding (hyperbola setting; GraphPad Prism version 4.00 for Windows, Graphpad Software, San Diego, California, USA, www.graphpad.com). The response units were recorded as the difference between the measuring and the reference cell. Note, the R26A and H33Q substitutions were numbered according to the consensus numbering; R82A and H89Q would be the actual numbering based on the ERj1 sequence.