Specification of Hsp70 Function by Type I and Type II Hsp40 - The Networking of Chaperones by Co-chaperones - page 97

C-terminal domain. Studies of the yeast Type II Hsp40, Sis1, have also localized
the polypeptide-binding site to CTDI (Sha et al. 2000 ; Lee et al. 2002 ). Therefore,
similar regions within Ydj1 and Sis1 are implicated in polypeptide binding.
Crystal structures of the C-terminal domains of both Ydj1 and Sis1 have been
solved (Fig. 4.2 ). These structures confirm that the C-terminal domain is a site for
peptide binding for both types of Hsp40s and they suggest similar yet unique mech-
anisms for substrate binding. The Ydj1 crystal structure is of the monomeric form
of a truncated C-terminal domain (Ydj1 102-350) in complex with a short peptide
substrate, GWLYEIS (Li et al. 2003 ; Li and Sha 2003 ). There are two hydrophobic
depressions, one in domain 1 and one in domain 3. The crystal structure shows that
the peptide substrate binds to Ydj1 by forming an extra ʲ-strand in the domain 1
depression. There is also an interaction in which the L from the peptide is buried in
a small hydrophobic pocket found in this surface depression. The pocket that the L
is buried in is formed by a variety of highly conserved hydrophobic residues (I116,
L135, L137, L216, and P249), thereby suggesting that the pocket may be a common
feature found in Type I Hsp40s, and may play a role in determining the substrate
specificity.
The X-ray crystal structure of Sis1 171-352 was also solved and it depicts a
homodimer that has a crystallographic two-fold axis (Sha et al. 2000 ; Qian et al.
2002 ). Sis1 171-352 monomers are elongated and constructed from two barrel-like
domains that have similar folds and mostly ʲ-structure. Sis1 dimerizes through a
short C-terminal α-helical domain, and the dimer has a wishbone shape with a cleft
that separates the arms of the two elongated monomers. CTDI on each monomer
also contains two shallow depressions that are lined by highly conserved solvent
exposed hydrophobic residues (Fig. 4.2 ). Mutational analysis of the residues that
line the hydrophobic depression in Sis1 has identified K199, F201 and F251 as
amino acids that are essential for cell viability and required for Sis1 to both bind
denatured substrates and cooperate with Hsp70 to refold those substrates (Lee et al.
2002 ; Fan et al. 2004 ). Interestingly, peptides from the C-terminal lid domain of
Hsp70 are also bound by in the hydrophobic depression on CTDI (Qian et al. 2002 ).
It is therefore possible that Hsp70 and substrates interact with Sis1 at similar sites.
If true, then Hsp70 might displace substrate from Hsp40s to drive substrate transfer
from Hsp40 to Hsp70 polypeptide binding domain (Kota et al. 2009 ; Summers et al.
2009a ).
Hsp40 Quaternary Structure
A common feature of Type I and Type II Hsp40s is that dimerization is important for
them to function in vivo (Summers et al. 2009a , b ). There are no crystal structures
of full length Type I or Type II Hsp40s, but small angle X-ray scattering (SAXS)
and protein modeling have been used to build models of the quaternary structure of
Type I and Type II Hsp40s (Borges et al. 2005 ; Ramos et al. 2008 ). These models
suggest that there are substantial differences in the quaternary structure of the Type