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. 2005 Mar 15;386(Pt 3):453-60.
doi: 10.1042/BJ20041050.

Structure-based mutagenesis studies of the peptide substrate binding fragment of type I heat-shock protein 40

Jingzhi Li  1 , Bingdong Sha
Affiliations

Structure-based mutagenesis studies of the peptide substrate binding fragment of type I heat-shock protein 40

Jingzhi Li et al. Biochem J. .

Abstract

Ydj1 is the major type I Hsp40 (heat-shock protein 40) family member in yeast. Ydj1 can pair with yeast Hsp70 Ssa1 to facilitate protein translocation and protein folding. Ydj1 itself can also function as a molecular chaperone to bind the non-native polypeptides and suppress protein aggregations in vitro. The crystal structure of Ydj1 complexed with its peptide substrate GWLYEIS reveals that a hydrophobic pocket located on Ydj1 domain I may play a major role in mediating the interactions between Ydj1 and the peptide substrate. To understand the mechanism by which Ydj1 interacts with non-native polypeptide, we have mutated the residues forming the hydrophobic pocket, based on the structural information. We have also constructed deletion mutations of the zinc-finger motifs within Ydj1. We have examined the functional consequences of these Ydj1 mutants by in vivo and in vitro assays. The results indicated that the hydrophobic pocket located on Ydj1 plays a critical role in its molecular chaperone activity by mediating interactions with the non-native polypeptides.

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Figures

Figure 1
Figure 1. The structure of Hsp40 Ydj1 complexed with the peptide substrate and the sequence alignment of the C-terminal regions from Hsp40 family members
(A) The ribbon drawing of the Ydj1 peptide-binding fragment complexed with the peptide substrate GWLYEIS. For Ydj1 structure, the α-helices are shown in light blue and the β-strands are shown in green. The bound peptide GWLYEIS is shown in blue. The two zinc-finger motifs are labelled. The two zinc atoms are shown as red spheres. The bar at the bottom of the Figure indicates 100 Å. (B) GRASP presentations of the Ydj1 and the peptide substrate complex structure. The orientation of the complex molecule in (B) is similar to that in (A). Blue and red denote positively and negatively charged regions respectively. The bound peptide GWLYEIS is shown as a rod model. The residues of the peptide substrate GWLYEIS are labelled in black. In the rod model, carbon atoms are shown in white, oxygen atoms are shown in red and the nitrogen atoms are shown in blue. The residues involved in forming the hydrophobic pocket are labelled in white. (C) Sequence alignment of the C-terminal regions from Hsp40 family members. Program Pileup from the GCG package was utilized to align the C-terminal peptide-binding fragment of type I Hsp40 Ydj1 from Saccharomyces cerevisiae with similar regions of type I Hsp40 proteins from Homo sapiens Hdj-2 and E. coli DnaJ. The zinc-finger motifs (CXXC) within type I Hsp40s are labelled in blue. The residues that are involved in forming the hydrophobic pocket on Ydj1 domain I are marked in red.
Figure 2
Figure 2. Molecular chaperone activities of Ydj1 mutants to assist Hsp70 Ssa1 in folding chemically denatured luciferase
The horizontal axis indicates the reaction time in minutes and the vertical axis shows the normalized refolded luciferase activities. Wild-type Ydj1 was used as the positive control and the refolded luciferase activity with wild-type Ydj1 and Hsp70 Ssa1 after 60 min reaction time is defined as 100% in this Figure. The refolded luciferase activity with no Ydj1 in the reaction is used as the negative control. (A) The molecular chaperone activities of the Ydj1 missense mutants. The residues involved in forming the hydrophobic pocket located on Ydj1 domain are mutated to alanine residues and their chaperone activities to assist Hsp70 Ssa1 to refold chemically denatured luciferase are measured. (B) The molecular chaperone activities of the Ydj1 zinc-finger motif deletion mutants to assist Hsp70 Ssa1 in folding the chemically denatured luciferase.
Figure 3
Figure 3. The Hsp70-independent molecular chaperone activities of Ydj1 mutants in suppressing aggregations of the chemically denatured substrates (luciferase and rhodanese)
The horizontal axis indicates the reaction time in min and the vertical axis shows the normalized light scattering values (D320). Wild-type (WT) Ydj1 was used as the positive control. The residues involved in forming the hydrophobic pocket located on Ydj1 domain are mutated to alanine residues and their independent chaperone activities to prevent chemically denatured luciferase (A) and rhodanese (B) aggregation are measured. (C) Binding of Ydj1 to unfolded luciferase measured by ELISA.
Figure 4
Figure 4. Growth phenotypes of ydj1 mutants
The wild-type or mutant ydj1 genes were transformed into the yeast strain Δydj1 by the plasmid shuffle method and selected on media without Uracil. Strains were grown at (A) 30 °C or (B) 37 °C for 4 days.
Figure 5
Figure 5. Western blotting of the yeast lysates reveals the expression levels of the yeast strains transformed with ydj1 mutants
The ydj1 wild-type (WT) and the mutants are labelled in the Figure.
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