title: The four hydrophobic residues on the Hsp70 inter-domain linker have two distinct roles.
abstract: The ubiquitous molecular chaperone 70-kDa heat shock proteins (Hsp70) play key roles in maintaining protein homeostasis. Hsp70s contain two functional domains: a nucleotide binding domain and a substrate binding domain. The two domains are connected by a highly conserved inter-domain linker, and allosteric coupling between the two domains is critical for chaperone function. The auxiliary chaperone 40-kDa heat shock proteins (Hsp40) facilitate all the biological processes associated with Hsp70s by stimulating the ATPase activity of Hsp70s. Although an overall essential role of the inter-domain linker in both allosteric coupling and Hsp40 interaction has been suggested, the molecular mechanisms remain largely unknown. Previously, we reported a crystal structure of a full-length Hsp70 homolog, in which the inter-domain linker forms a well-ordered β strand. Four highly conserved hydrophobic residues reside on the inter-domain linker. In DnaK, a well-studied Hsp70, these residues are V389, L390, L391, and L392. In this study, we biochemically dissected their roles. The inward-facing side chains of V389 and L391 form extensive hydrophobic contacts with the nucleotide binding domain, suggesting their essential roles in coupling the two functional domains, a hypothesis confirmed by mutational analysis. On the other hand, L390 and L392 face outward on the surface. Mutation of either abolishes DnaK's in vivo function, yet intrinsic biochemical properties remain largely intact. In contrast, Hsp40 interaction is severely compromised. Thus, for the first time, we separated the two essential roles of the highly conserved Hsp70 inter-domain linker: coupling the two functional domains through V389 and L391 and mediating the interaction with Hsp40 through L390 and L392.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Divya Prasanna
authors.initials: DP
authors.lastname: Kumar
authors.forename: Christina
authors.initials: C
authors.lastname: Vorvis
authors.forename: Evans Boateng
authors.initials: EB
authors.lastname: Sarbeng
authors.forename: Vanessa C
authors.initials: VC
authors.lastname: Cabra Ledesma
authors.forename: John Eric
authors.initials: JE
authors.lastname: Willis
authors.forename: Qinglian
authors.initials: Q
authors.lastname: Liu
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D018832:Molecular Chaperones; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D020349:Surface Plasmon Resonance
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D000251:Adenosine Triphosphatases