Dissection of the ATP-binding domain of the chaperone hsc70 for interaction with the cofactor Hap46

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.