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Characterization of the effect of naturally occurring mutations on the chaperone
function of the human HSPA1A.
Peter Nguyen, Andrei Bilog, and Nikolas Nikolaidis
Department of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, California State University Fullerton, Fullerton, CA
 Background
 Results
Seventy-kDa heat shock proteins (Hsp70s) are molecular chaperones
involved in protein folding, synthesis, and transportation across
STRESS
membranes.
I480N
ADP
9.00E-04
*
4.00E-05
F592S
native
protein
ref olds
3
unf olds
translation
binds to
Hsp70
endoplasmic
reticulum
binds to
1
1
misf olded
protein
1
refolds
stress-denatured or
nascent chain peptides
2
mitochondria
p= 0.049
3.50E-05
1.00E-04
Figure 1:
Classical cell
stress response
pathway
mediated by
Hsp70s.
2. Transporting proteins
across membranes
p= 0.003
8.00E-05
p= 0.001
*
*
p= 0.003
6.00E-05
p= 0.004
6.00E-04
p= 0.045
2.50E-05
*
p= 0.04
5.00E-04
2.00E-05
4.00E-04
1.50E-05
3.00E-04
1.00E-05
2.00E-04
5.00E-06
1.00E-04
*
*
*
4.00E-05
2.00E-05
ATPase
Domain
prevents aggregation
3. Regulating cell
survival
0.00E+00
0.00E+00
1
1
WT
I74T
S16Y
I74T
I480N
F592S
0
WT
S16Y
I74T
F592S
WT
S16Y
I74T
I480N
40
F592S
p= 0.0003
1
*
p= 0.001
-10
p= 0.0006
20
Figure 4: Three-dimensional representation of the HSPA1A protein molecule showing the
position of the mutations. The ATPase domain (blue) and substrate-binding domain (green)
are connected by the hydrophobic linker (red). Inlets show a magnification of the WT amino
acid residues (yellow) and the mutations (magenta).
dS (J/mol*K)
-20
Hsp70s function by binding and releasing unfolded or misfolded
polypeptides in an ATP depending manner.
0
-30
C
1
-40
-40
-20
-50
-60
-40
*
-60
p= 0.048
-80
-70
-60
*
-100
p= 0.01
B
*
*
-20
-80
A
I480N
1
0
1
S16Y
Figure 2: The Hsp70 has
dual characteristics intrinsic function as an
ATPase and also interaction
with co-chaperones such as
the J protein as well as the
nucleotide exchange
factors.
7.00E-04
*
3.00E-05
0.00E+00
3
1. Maintaining of protein
homeostasis
native
protein
8.00E-04
1.20E-04
Kd (M)
Nucleus
Substrate-binding
Domain
p= 0.001
*
HSF
modulates
anti-inflammatory
effectors
stimulates
pro-inflammatory
cytokines
p= 0.03
4.50E-05
1.40E-04
inhibits apoptotic
pathways
peptide
ATP
-80
-90
-100
-100
-120
WT
S16Y
I74T
I480N
F592S
0
WT
S16Y
I74T
I480N
WT
F592S
0
S16Y
I74T
I480N
F592S
0
1
1
1
-5
-10
-10
dH (kJ/mol)
-20
WT
HSPA1A is the major heat-inducible Hsp70 in humans and is a key
player in several signaling pathways that regulate protein
homeostasis, cell survival. This protein has been associated with a
variety of human conditions including breast and ovarian cancer,
atherosclerosis, and Alzheimer’s disease. Therefore, it is important
to determine functional changes of this protein as they may be
related with several conditions and diseases.
-10
-30
 Methods
Clone wildtype (WT) and
mutant
HSPA1A
Purify
recombinant
proteins from
bacteria
Determine functional changes
by Isothermal Titration Calorimetry
(ITC)
F592S
I480N
The goal of this project was to determine the functional output of
natural occurring mutations (SNPs) on the HSPA1A gene in different
human populations .
Figure 5: ITC analysis of WT and mutated HSPA1A variants with ADP(A), ATP (B), and protein
substrate (C).
p= 0.047
-40
-30
-40
-35
-50
-40
*
-50
-60
-45
-70
WT
I74T
 Purpose
*
-25
-60
S16Y
Figure 3: Single nucleotide
polymorphisms (SNPs) are differences
in one nucleotide base in a DNA
sequence.
-20
-30
p= 0.004
In humans, a major source of genetic variation results from single
nucleotide polymorphisms (SNPs); they may or may not alter the
encoded amino acid.
-15
-20
S16Y
I74T
I480N
F592S
-50
WT
S16Y
I74T
I480N
F592S
WT
S16Y
I74T
I480N
F592S
Figure 6: Statistical analysis of WT compared to the mutant HSPA1A variants. Isothermal titration
calorimetry revealed that the I480N mutant differs significantly in its affinity for ADP, ATP, and
peptide substrate. This mutant also displayed significant different reaction entropy as compared
to the WT HSPA1A (N=4; bars= S.D.; p values are the results of a student’s t-test). The S16Y
mutant differs significantly in its affinity to both ADP and ATP. This mutant also displayed
different reaction entropy and enthalpy from WT HSPA1A. N=3; bars= S.D.; p-values are the
results of a student’s t-test.
 Conclusions
- Particular mutations significantly alter the binding affinity to both nucleotide and
protein substrates.
- Some mutations resulted in changes in the entropy and enthalpy of the reactions,
revealing alterations in the way the molecules interact and the reaction products are
formed.
- Natural mutations alter the function of HSPA1A.
 Future Work
- Test in vivo whether the observed functional changes are biologically important.
 Acknowledgements
This work was supported by a State Mini-Grant to NN. We would also like to thank our lab mates in Nikolaidis lab as well
as Dr. Rasche for allowing us to use the ITC machine.