Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

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.The ubiquitously distributed Hsp70 family of molecular chaperones shepherd newly synthesized polypeptide chains, protect cells from stress-induced protein aggregation, assist in protein translocation across membranes, and regulate assembly and disassembly of macromolecular complexes. These physiological functions are accomplished by a two-domain allosteric mechanism in which cycles of ATP binding and hydrolysis in the N-terminal nucleotide-binding domain (NBD) 2 control the binding and release of hydrophobic polypeptide segments in the substrate-binding domain (SBD) (1-3). Although we have gained an increasingly detailed picture of this allosteric transition in Hsp70 chaperones and modes of substrate interaction (4 -7), it is striking that there is much less known about the function of the extreme C-terminal unstructured region (Fig. 1). Binding of the C-terminal region of mammalian Hsc70 to substrate was suggested in earlier work (8, 9), and more recently, the C-terminal segment of eukaryotic cytoplasmic Hsp70s was found to contain a conserved tetratricopeptide repeat (TPR) domain interaction motif that mediates binding with Chip, Hip, or Hop co-chaperones that facilitate the assembly of a variety of Hsp70 complexes (3). Even though bacteria lack homologs of these Hsp70-interacting TPR domain proteins, many, including the extensively characterized E. coli Hsp70 DnaK, have a disord...

BiP and Its Nucleotide Exchange Factors Grp170 and Sil1: Mechanisms of Action and Biological Functions

“…In this case, direct interactions between the lid and the substrate were observed, leading to even further extension of the conformational space available within the Hsp70s’ SBD. This direct lid-substrate interaction, as well as the recently described interaction between the unstructured C-terminus of Hsp70 chaperones and their substrates 64 , might aid in conformational remodeling of Hsp70 clients. The ATP-induced opening of the lid 51; 52 and the inherent dynamics observed for the lid 48; 57 might also have an impact on substrate conformations if lid-substrate interactions occur.…”

How Hsp70 Molecular Machines Interact with Their Substrates to Mediate Diverse Physiological Functions

“…They showed that a relatively short truncation (35 residues) led to loss of viability under stringent conditions (absence of SecB) and loss of ability to efficiently refold denatured luciferase in vitro . 36 In addition, the C-terminus of E. coli DnaK became more ordered when DnaK bound to a protein model substrate (a stably unfolded staphylococcal nuclease mutant) but not when bound to a peptide. 36 Taken together these studies suggest that protein substrates may bind to the C-terminus of Hsp70s in addition to the canonical substrate-binding groove.…”

“…36 In addition, the C-terminus of E. coli DnaK became more ordered when DnaK bound to a protein model substrate (a stably unfolded staphylococcal nuclease mutant) but not when bound to a peptide. 36 Taken together these studies suggest that protein substrates may bind to the C-terminus of Hsp70s in addition to the canonical substrate-binding groove. This type of binding may serve multiple purposes–keeping the substrate more unfolded and also keeping the substrate in the local region of the chaperone even after ATP-mediated release, to facilitate rebinding if necessary.…”

Conditional disorder in chaperone action

“…The C terminus of Hsp70 is also disordered, but its exact role in chaperone activity remains controversial. Smock et al presented evidence that the C terminus of Hsp70 encodes a weak auxiliary peptide binding motif that is important for chaperone activity in vitro [43]. However, Aponte et al performed an in vivo selection for enhanced chaperone activity and found that truncation of 35 residues from the disordered C terminus of Hsp70 actually increased in vivo and in vitro chaperone activity [44].…”

“…However, Aponte et al performed an in vivo selection for enhanced chaperone activity and found that truncation of 35 residues from the disordered C terminus of Hsp70 actually increased in vivo and in vitro chaperone activity [44]. One explanation for these apparently conflicting results is that chaperones bind their own disordered regions and thus might treat themselves as substrates [43]. Disordered regions in chaperones like Hsp70 would thus be acting as covalently linked inhibitors.…”