HSP40: Figures

Figure 1. Structural classification of DNAJ/HSP40 family members.

HSP40 Structure - Structural classification of DNAJ/HSP40 family members.

Figure 1. Structural classification of DNAJ/HSP40 family members.

Depending on the presence of the Gly/Phe (G/F)-rich linker region and/or the Cys-rich zink finger (ZF) domain, DNAJs/HSP40s can be grouped into three subtypes. All DNAJs/HSP40s harbor the characteristic J-domain containing a highly conserved HPD motif. In type 1 and type 2 family members, the J-domain is located at the N-terminus while in type 3 family members it can be found at any position within the molecule. Type 1 proteins are similar to E. coli DnaJ with a G/F-rich region adjacent to the J-domain followed by the ZF and C-terminal peptide-binding domain (CTD) composed of two barrel topology subdomains, CTD-1 and CTD-2, and a dimerization domain (DD) at the extreme C-terminus. Type 2 family proteins lack the ZF motifs, while typ3 family members possess only the J-domain together with the DD.

 

Figure 2. Structures of the J-domain in the human type 2 DNAJ/HSP40 family member DnaJB1/Hdj1.

Hsp40 Structure - Structures of the J-domain in the human type 2 DNAJ/HSP40 family member DnaJB1/Hdj1.

Figure 2. Structures of the J-domain in the human type 2 DNAJ/HSP40 family member DnaJB1/Hdj1 (PDB ID: 1HDJ) 52.

(A) The J-domain (residues 1-76) is composed of four helices and a loop region between helix II and III bearing the highly conserved HPD motif. (B) Electrostatic surface distribution of the DnaJB1/Hdj1 J-domain. Blue and red colors of the electrostatic surface diagram correspond to positive and negative electrostatic potentials of standard residues in proteins and polynucleotides. Similarly, electro-positive and electro-negative polar atoms are skyblue and salmon, respectively. Default element colors are: carbon (grey), nitrogen (light blue), oxygen (light pink), sulphur (green), phosphorus (olive-green), selenium (magenta). (C) NMR ensemble of the DnaJB1/Hdj1 J-domain. The NMR ensemble consists of five conformers.

 

Figure 3. Crystal structure of the putative peptide-binding fragment in human DnaJB1/Hdj1.

HSP40 Structure - Crystal structure of the putative peptide-binding fragment in human DnaJB1/Hdj1.

Figure 3. Crystal structure of the putative peptide-binding fragment in human DnaJB1/Hdj1 (PDB ID: 2QLD) 176.

In the crystal structure, the putative peptide-binding fragments associate to form a homodimer via their C-terminal dimerization motifs. The DnaJB1/Hdj1 homodimer possesses a U-shaped architecture with a large cleft constituted between the two elongated monomers (N- and C-termini are labelled accordingly).

 

Figure 4. Chaperone Hsp70 and its co-chaperone Hsp40.

HSP40 Structure - Crystal structure of Hsp70 and its co-chaperone Hsp40.

Figure 4. Chaperone Hsp70 and its co-chaperone Hsp40.

Cartoon illustrating the (a) ATP-open conformation (PDB ID: 4B9Q; 417) and the (b) ADP-bound closed conformation (PDB ID: 2KHO; 418) of E. coli Hsp70 DnaK. The nucleotide binding domain is given in red, the substrate binding domain in blue, and the C-terminal lid in cyan. (c) Ribbon and tube representation of the tertiary structure of the C-terminal homodimeric DnaJB1/Hdj1 peptide-binding fragment (blue) complexed with the Hsp70 C-terminal EEVD peptide (magenta, PDB ID: 3AGY; 203). (d) Structure of the J-domain of yeast DnaJ/Hsp40 Sis1 (red; PDB ID: 4RWU). The dashed line indicates the connectivity of DnaJ/Hsp40 domains (reprinted from Clare and Saibil 200).

 

Figure 5. Proposed model of DnaJ/Hsp40-dependent substrate binding to Hsp70/DnaK.

HSP40 Function - DnaJ/Hsp40-dependent substrate binding to Hsp70/DnaK

Figure 5. Proposed model of DnaJ/Hsp40-dependent substrate binding to Hsp70/DnaK.

In the ATP-bound state, the nucleotide binding domain (NBD; also known as ATPase domain) and substrate binding domain (SBD) are docked, the substrate binding groove is open, and the Hsp70/DnaK chaperone exhibits low affinity and fast exchange rates for its substrate. ATP hydrolysis is facilitated by J-domain proteins (JDPs) of the DNAJ/HSP40 family and nucleotide exchange by nucleotide exchange factors (NEFs) such as GrpE and Bag-1. DNAJs/HSP40s function as homodimers and bind to unfolded or non-native polypeptides (Su) via their C-terminal SBD in order to prevent aggregation. DnaJ/Hsp40-bound unfolded substrate is delivered subsequently to Hsp70/DnaK. DnaJ/Hsp40 then interacts with the N-terminal ATPase domain of Hsp70/DnaK. The simultaneous interplay of the substrate with the SBD of Hsp70/DnaK and of the JDP with the NBD of Hsp70/DnaK induces a conformational change in the ATPase domain thereby stimulating ATP hydrolysis and mediating the closure of the substrate binding groove. In the ADP-bound state, the docking between the two domains of Hsp70/DnaK is interrupted and the chaperone exhibits high affinity and low exchange rates for its substrate. ADP release is mediated by the specific interaction of NEFs with the Hsp70/DnaK ATPase domain followed by a conformational change which results in a low-affinity state and release of the substrate. The released substrate can be either folded into the native protein (Sn), re-bound to Hsp70/DnaK or aggregate (Sa).

 

Figure 6. Inhibitors of DnaJ/Hsp40.

HSP40 Inhibitors - Quercetin, KNK437, butyl 3-[2-(2,4-dichlorophenoxy)acetamido]benzoate

Figure 6. Inhibitors of DnaJ/Hsp40.

Quercetin (3,3′,4′,5,7-pentahydroxyflavon) suppresses the expression of HSPs (e.g. DnaJ/Hsp40) by depleting cellular stores of HSF-1 and inhibits the acquisition of thermotolerance in cancer cells 413. Quercetin also promotes dimerization of the neuroprotective DnaJC5/Csp-a 415. The benzylidene lactam compound, KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-╬│butyrolactam) inhibits HSP expression and the acquisition of thermotolerance more potently than quercetin 414. The phenoxy-N-arylacetamide derivative butyl 3-[2-(2,4-dichlorophenoxy)acetamido]benzoate binds directly to DnaJ/Hsp40 and functions as an effective inhibitor 416.