Abstract
The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide- and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.
Highlights
To explore the functional significance of this C-terminal region, we carried out a comparative sequence analysis of Hsp70 family members, and two large and distinct groups emerged based on patterns of C-terminal conservation: those containing the eukaryotic tetratricopeptide repeat (TPR) domain interaction motif and those harboring a previously undescribed C-terminal motif shared among diverse bacterial sequences, including Escherichia coli DnaK, and several eukaryotic organellar Hsp70s
Another cluster (221 sequences colored yellow in Fig. 2A) is composed of Hsp70 with very weakly homologous C termini, such as one subgrouping of sequences with a partial internal deletion culminating in the tripeptide DEL or a similar motif, as exemplified by E. coli HscA
One of the clusters (157 sequences colored blue in Fig. 2A) is composed of strictly eukaryotic Hsp70s that culminate in an EEVD sequence motif for TPR domain interaction, including human paralogs Hsc70 and Hsp70-1
Summary
To explore the functional significance of this C-terminal region, we carried out a comparative sequence analysis of Hsp70 family members, and two large and distinct groups emerged based on patterns of C-terminal conservation: those containing the eukaryotic TPR domain interaction motif and those harboring a previously undescribed C-terminal motif shared among diverse bacterial sequences, including Escherichia coli DnaK, and several eukaryotic organellar Hsp70s. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.
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