GrpE, Hsp110/Grp170, HspBP1/Sil1 and BAG Domain Proteins: Nucleotide Exchange Factors for Hsp70 Molecular Chaperones - The Networking of Chaperones by Co-chaperones - page 4

the ATP hydrolysis rate of DnaK substantially. The spontaneous transition between
the two states is slow as Hsp70 has intrinsically only weak ATPase activity. This
prevents substrate-free cycling. The cycle is reset with the release of ADP and re-
placement with ATP, which releases the client protein for a new folding attempt.
DnaK, DnaJ and GrpE: The Eubacterial Hsp70 System
For its proper functioning in protein folding, DnaK is dependent on the ATPase-
stimulating cochaperone DnaJ and the nucleotide exchange function of GrpE
(Fig. 1.1a ). Although interactions with substrate protein trigger ATP hydrolysis in
DnaK, meaningful folding rates with model proteins are only achieved in presence
of DnaJ, the prototypical Hsp40 protein (Laufen et al. 1999 ). Hsp40 and other J-do-
main proteins are reviewed in Chapter 4. Because of DnaK's slow off-rate for ADP,
additional presence of GrpE is essential for E. coli cells to reset the Hsp70-folding
cycle (Ang and Georgopoulos 1989 ). The combined action of the two cofactors is
thought to drive the folding cycle of the molecular chaperone, resulting in repetitive
rounds of substrate binding and release.
GrpE functions as the nucleotide exchange factor for DnaK by stabilizing a
NBD conformation with an open nucleotide binding cleft (Harrison et al. 1997 )
(Fig. 1.1a ). The crystal structure revealed that subdomain IIB of DnaK is rotated
outwards in the complex, which weakens the contacts to ADP (Fig. 1.2 ).
The E. coli cytosol comprises of two additional isoforms of Hsp70, HscA and
HscC, and five more proteins containing a J-domain. These isoforms and their as-
sociated J-protein cofactors have more specialized functions than DnaK, such as
incorporation of Fe-S clusters into substrates using the IscU scaffold protein. In con-
trast, DnaK appears to be the more general-purpose protein-folding machine. Inter-
estingly, functioning of HscA does not require the NEF GrpE (Brehmer et al. 2001 ).
The Evolution of Eukaryotic Hsp70 Systems
In eukaryotes, close sequence homologs to GrpE are only found in mitochondria
and chloroplast, i.e. organelles of eubacterial origin, whereas orthologs to DnaK
and DnaJ are found in the cytosol/nucleus and the ER lumen. These endosymbiont-
derived organelles have thus preserved an eubacterial protein folding machinery
(homologs to GroEL, GroES, HtpG and ClpA are further evidence for this), al-
peptide backbone is shown in ribbon representation, and the bound nucleotide as space-filling
model (PDB code 4B9Q (Kityk et al. 2012 )). The nucleotide binding, ʲ-sandwich and α-helical
domains are indicated in blue , green and yellow , respectively. c NMR model for the DnaKﾷADP
complex. In this state, the NBD and SBD are loosely associated (PDB code 2KHO (Bertelsen et al.
2009 )). The representation mode is the same as in b . The peptide NRLLLTG from the complex
structure with the SDB alone (PDB code 1DKZ (Zhu et al. 1996 )) is superposed