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Dissection of the ATP-binding domain of the chaperone hsc70 for interaction with the cofactor Hap46
G Petersen1, C Hahn, U Gehring
1Institut für Molekulare Genetik, Im Neuenheimer Feld 230 and Biochemie-Zentrum Heidelberg, Biologische Chemie, Im Neuenheimer Feld 501, Universität Heidelberg, D-69120 Heidelberg, Germany. gabi@sirius.mgen.uni-heldelberg.de
Insights
Hap46 (BAG-1) interacts with the ATP-binding domain of heat shock protein 70 (hsp70) and hsc70, influencing their chaperone functions. This study identifies key contact sites on hsp70/hsc70 involved in this crucial molecular interaction.
Area of Science:
- Molecular Biology
- Protein Interactions
- Chaperone Proteins
Background:
- Mammalian hsp70 chaperone proteins are involved in various cellular processes.
- Hap46 (BAG-1) is a protein that interacts with hsp70/hsc70 independently of the substrate-binding site.
- This interaction affects the nucleotide-binding and protein-folding properties of hsp70/hsc70.
Purpose of the Study:
- To identify the specific contact sites on hsp70/hsc70 involved in the interaction with Hap46.
- To understand how Hap46 binding influences the conformation of the hsp70/hsc70 ATP-binding domain.
Main Methods:
- Screening of membrane-bound peptide libraries based on the ATP-binding domain sequence of hsp70/hsc70.
- Phage-display technique using random dodecapeptides to identify interaction regions.
- Analysis of identified contact sites on a space-filling model of hsc70.
Main Results:
- Both peptide library screening and phage display identified partially overlapping contact regions.
- Two major contact areas for Hap46 were delineated on the hsc70 molecule.
- These regions are located on either subdomain bordering the nucleotide-binding cleft.
Conclusions:
- The interaction of Hap46 with hsp70/hsc70 involves specific contact sites on the ATP-binding domain.
- These binding sites bridge the subdomains around the nucleotide-binding cleft.
- This interaction likely alters the conformation of the ATP-binding domain, similar to GrpE's effect on DnaK.
Abstract:
Several unrelated proteins are known that specifically interact with members of the mammalian hsp70 chaperone protein family independent of the hsp70 substrate-binding site. One of these is Hap46, also called BAG-1, which binds to the ATP-binding domain of hsp70 and its constitutively expressed, highly homologous counterpart hsc70, thereby affecting nucleotide binding, as well as protein folding properties, of these molecular chaperones. In an attempt to delineate the potential contact sites on hsp70/hsc70 involved in this interaction we made use of the following two independent approaches: (i) screening of membrane-bound peptide libraries based on the sequence of the ATP-binding domain and (ii) the phage-display technique with random dodecapeptides. These approaches yielded partially overlapping results and identified several possible contact regions. On the space-filling model of hsc70, the two major contact areas for Hap46 delineated in the present study are located on the same side of the molecule on either subdomain that border the central cleft harboring the nucleotide-binding site. We suggest that this bridging affects the conformation of the ATP-binding domain in a way similar to the opening of the nucleotide-binding cleft produced in the bacterial hsp70 homologue DnaK upon binding its regulatory protein GrpE.