Biomedical Engineering Reference
In-Depth Information
models can explain the data. The inferred presence of an external binding Cd
2+
site leads to a model of how an external binding site may be essential to the
Cd
2+
binding process to the central site.
10.5 Binding of a 10 kDa Ligand to a 70 kDa Protein
Does not Result in Significant Line Broadening of
the NMR Signals of the 10 kDa Ligand
The Hsp70/Hsp40 proteins (heat shock proteins 70 and 40 kDa), form an
essential
chaperone
system
that
facilitates
the
folding
and
re-folding
of
proteins in stressed and un-stressed cells.
22
The proteins are upregulated in
tumours,
23
and are involved in Alzheimer's disease.
24
In bacteria the Hsp70/
Hsp40 system is called DnaK/DnaJ.
DnaK is an allosteric protein. When ADP is bound to the 45 kDa nucleotide
binding domain (NBD), the 25 kDa substrate binding domain (SBD) is
undocked,
25
and binds substrate with high affinity. When ATP is bound, the
NBD and SBD are docked leading to substrate release.
22
The allosteric cycle is aided by the co-factors DnaJ and GrpE.
22
DnaJ
recruits misfolded substrates to the DnaK SBD. Subsequently DnaK
hydrolyses ATP. This leads to a large-scale conformational change and the
substrate becomes more tightly bound. An active unfolding of the substrate
proceeds by a process called 'entropic pulling'.
27
The nucleotide-exchange
factor GrpE allows back-exchange of ATP, reducing the affinity for the now
unfolded substrate. The unfolded substrate is released and can refold.
DnaJ is a 40 kDa, multi-domain protein, containing an N-terminal 70-
residue J-domain, followed by a Gly/Phe-rich region, a Zn-Cys domain, a
substrate binding domain and a dimerisation domain. The J-domain alone is
sufficient to stimulate the ATPase activity of DnaK.
28
The J-domain is an anti-parallel two-helix bundle, referred to as helices
II
and
III
, with two small adjacent helical elements as determined by NMR.
29,30
The GF-region (residues 71-108) is dynamic and disordered.
30
A crystal
structure is available for a YDJ1(110-337) dimer, a yeast protein homologous
to DnaJ.
31
Here we describe the use of NMR to characterise the binding of the J-
domain (10 kDa) to full-length DnaK (70 kDa).
32
10.5.1 A View From the DnaJ Perspective
When following a
15
N-
1
H HSQC-TROSY of 60 mM
15
N-labelled J-domain on
addition of unlabelled DnaK, small chemical shift changes are observed
[Figures 10.15 and 10.16(a)]. The affected residues form a contiguous region
[Figure 10.16(b)] (helix
II
) and the binding saturates with a stoichiometry of
1 : 1 and a K
D
of 16 mM [Figure 10.17(a)]. These results were in agreement with
earlier work of others.
33
