Sequence-specific control of ETS transcription factors: cause and effects June 7, 2012 Department of Chemistry Georgia State University Gregory Poon Department of Pharmaceutical Sciences College of Pharmacy Washington State University
Sequence-specific control ofETS transcription factors: cause and effects
June 7, 2012Department of ChemistryGeorgia State University
Gregory PoonDepartment of Pharmaceutical Sciences
College of PharmacyWashington State University
From: HHMI, Duke University
ETS family of transcription factors
• Originally identified in transforming gene of an avian erythroblastosis virus, E26 ets = “e26 transformation specific”
• v-ets led to discovery of cellular homolog c-ets-1
• 28 known human ETS-related genes to-date
• Defined by a structurally conserved DNA-binding domain = ETS domain
Hollenhorst et al. (2011) Annu Rev Biochem 80, 437-471
Degnan et al. (1993) Nucleic Acids Res 21, 3479-3484
ETS proteins are widely distributed among animals (metazoa)
• Not found in plants, fungi, protoza
ETS is a winged helix-turn-helix domain
H1 H2
turn or “loop”
“wing”
H3
turn
H2
“wing”
Wei et al. (2010) EMBO J 29, 2147-2160.
Sequence selectivity among ETS proteins
Class III
Class I
Class II
Class IV
mEts-1 (1K79)
mElf-3 (3JTG)
mPU.1 (1PUE)
hPDEF (1YO5)
hFLI-1 (1FLI) hELK-1 (1DUX)
hSAP1-1 (1BC7)
mGABPα/β (1AWC)
PU.1, a model ETS protein• PU.1 is cellular homolog of viral oncogene Spi-1
Restricted to cells of hematopoietic and derived lineages Blood cell development, immune function, leukemia Regulates (mostly activates) expression of >50 genes
• Contains a minimal ETS domain (class III)• What is the physical basis of sequence selection?
Wei et al. (2010) EMBO J 29, 2147-160Szymczyna and Arrowsmith (2000) 37, 28363-379Poon, unpublished results
In vitro
in vivo: promoters
in vivo: ChIP-seq
Poon and Macgregor (2003) J Mol Biol, 328, 805-19Poon (2012) J Biol Chem 287, 18297-307
PU.1 ETS is a broadly sequence-selective protein
XXXGGAAYYY
AGCGGAAGTG
AAAGGAAGTG
AAAGGAATGG
AGCGAGAGTG
KD, nM
0.53 ± 0.13
2.68 ± 0.41
240 ± 70
1000
G°, kJ/mol
-4.03 ± 0.72
—
11.14 ± 0.81
14
High affinity
High affinity
Low affinity
Nonspecific
Selection of ETS binding sites
Y YYGXX GA X AAAA
CT X' C TTG G
TT
C C
Y'X'X' Y' Y'
GA
T C
AA
TT
A AA
TTTA
T
H(duplex), kJ/mol
338
312
314
299
Variants of B motif of Ig2-4 enhancer
PU.1 ETS-DNA interactions I:Structure
Sequence-specific ETS-DNA interactions
Indirect readoutProtein-phosphate contactsProtein-sugar contacts
Indirect readoutProtein-phosphate contacts
H3
turn
H2
“wing”
Direct readoutProtein-base contactsProtein-water-base contacts
Kodandapani et al. (1996) Nature 380, 456-460.
Arg235
Asn236
Arg232
T23
G9
G8
C26
C25
T24
Glu228
Lys229
5’-AAAAGGAAGTGGG-3’3’-TTTTCCTTCACCC-5’
H3
S205
K208
L250
Y252
K245
R235
5’-AAGGGGAAGTGGG-3’3’-TTCCCCTTCACCC-5’
“wing”
A
A
A
K223
N221
K219
W215
I172
R173
L174
5’-AAAAGGAAGTGGG-3’3’-TTTTCCTTCACCC-5’
H3
H2
turn or “loop”
T
T
C
A
Characterization of the protein-DNA interface in solution: footprinting
Single-hit conditions
* *
Cleavage efficiency depends on:Chemistry
AccessibilitySize of reagent
Solvent exposure of target
0.01 0.1 1 10
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
single cut
Poi
sson
pro
babi
lity
Average cut per strand
uncut
multiple cuts
KMnO4
OsO4
·OH (sugar) ·OH (sugar) ·OH (sugar)
DEPCDMS/piperidine
Biochemical probes of nucleic acids
Phosphates: DNase I, UO22+
DMS/NaOH
5’-C
TTG
GTT
TCA
CTT
CC
GC
TTAT
TT-3
’
3’-TTTATTTTCC
TTAC
CTTTG
GTTC
-5’
3’
5’
C+T DNase I protection: TTCC strand
-15 -11 -10 -9 -8 -7 -6 -50
500
1000
1500
2000
2500
3000
Inte
nsity
, AU
log [ETS, M]
-11.0-10.9 -10 -9 -8 -7 -6 -5T
T
T
A
T
T
T
T
C
C
T
T
A
C
C
T
log [ETS, M]
0.000
220.0
440.0
660.0
880.0
1100
1320
1540
1760
1980
2200
-15 -11 -10 -9 -8 -7 -6 -50
500
1000
1500
2000
Inte
nsity
, AU
log [ETS, M]
-15 -11 -10 -9 -8 -7 -6 -5
500
1000
1500
2000
2500
Inte
nsity
, AU
log [ETS, M]
High-affinity sequence
Low-affinity sequence
-10.9 -10 -9 -8 -7 -6 -5T
T
T
A
T
T
C
G
C
C
T
T
C
A
C
T
T
T
T
A
T
T
C
G
C
C
T
T
C
A
C
T
log [ETS, M]
0.000
290.0
580.0
870.0
1160
1450
1740
2030
2320
2610
2900
-15 -11 -10 -9 -8 -7 -6 -5
500
1000
1500
2000
2500
Inte
nsity
, AU
log [ETS, M]
-log [ETS, M]
Poon (2012) J Biol Chem 51, 18297-307
5’-AAAAGGAAGTGGG-3’3’-TTTTCCTTCACCC-5*’
5’-AAGGGGAAGTGGG-3’3’-TTTTCCTTCACCC-5’
Fiber model: Chandrasekaran & Arnott. (1989) The Landolt-Börnstein Database, DOI: 10.1007/10384901_24
ETS-induced DNA bending
5’-AAGGGGAAGTGGG-3’3’-TTCCCCTTCACCC-5’
-10
0
10
20
30
40
AA/TTAA/TT
AA/TT
AG/CT
GG/CC
GG/CC
GG/CC
GA/TCAA/TT
AG/CT
GT/AC
TG/C
A
GG/CC
GG/CC
4
6
8
10
12
14
16
tw
ist,
°
V
ALL,
VTI
L,
V
RO
L, °
cannonical
wid
th, Å
:
min
or g
roov
e,
maj
or g
roov
e
Poon (2012) Biochemistry 51, 4096-107
0.0 0.2 0.4 0.6 0.8 1.0
0.70
0.71
0.72
0.73
0.74
0.75
0.76
0.77
Rf(b
ound
) : R
f(free
)Flexure displacement
Poon, unpublished results
free
bound
Θ
AGCGGAAGTG Θ = 36°
Circular permutation analysis of DNA curvature
C+T DNase I protection: nonspecific sequence
GGAA strand TTCC strand
3’-TTTATTTTCTC
TAC
CTTTG
GTTC
-5’
-log [ETS, M]5
10 105
5’-*
AA
ATA
AA
AG
AG
ATG
GA
AA
CC
AA
G-3
’
0 2 4 6 8 10
0
10
20
30
40
50
Background
Bou
nd [3
2P-la
bele
d N
S o
ligo]
, pM
[NS oligo], µM
+ETS (50 nM)
KD = 1.7± 0.2 µM
Poon (2012) J Biol Chem 287, 18297-307
3’-TTTATTTTCC
TTAC
CTTTG
GTTC
-5’
DNase Ifootprinting
Gel mobilityshift
[salmon sperm DNA](mM bp)
C+T1 0.60
0.8 0.4 0.2
unbound
1:1 bound
Poon, unpublished results
0.0 0.2 0.4 0.6 0.8 1.0
0
1
2
3
4
5
6
7
Nor
mal
ized
hyp
erse
nsiti
vity
[Salmon sperm DNA], mM bp
·OH footprinting5’
-CTT
GG
TTTC
AC
TTC
CG
CTT
ATTT
-3’
-10.0 -9.3 -10.0 -9.3 -8.7 -8.0 -7.3 -6.7GCTCTAGATTTATTCGCCTTCACTTTGGTT3'
log [ETS, M]
0.3000
0.4450
0.5900
0.7350
0.8800
1.025
1.170
1.315
1.460
1.605
1.750
5'
1E-15 1E-10 1E-9 1E-8 1E-7 1E-6 1E-5
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Nor
mal
ized
inte
nsity
, AU
[ETS], M
High-affinity sequence
Poon (2012) Biochemistry 51, 4096-107
5’-C
TTG
GTT
TCC
ATT
CC
TTTT
ATTT
-3’
Low-affinity sequence
-10.0 -9.3 -8.7 -8.0 -7.3 -6.7 -6.0 -5.3GCTCTAGATTTATTTTCCTTACCTTTGGTTC
log [ETS, M]
0.2000
0.3700
0.5400
0.7100
0.8800
1.050
1.220
1.390
1.560
1.730
1.900
1E-15 1E-10 1E-9 1E-8 1E-7 1E-6 1E-50.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Nor
mal
ized
inte
nsity
, AU
[ETS], M
Poon (2012) Biochemistry 51, 4096-107
Sequence-specific binding induces local conformational changes in DNA
G T+8
T+7
T+6
C A C T+2 T+1 C C G C T-4
T-5
A T-7
T-8
T-9
0.00.20.40.60.81.01.21.41.61.8
-3'
-3'
5'-
Cle
avag
e re
lativ
e to
unb
ound
sta
te
AGCGGAAGTG
G T+8
T+7
T+6
C C A T+2 T+1 C C T-2
T-3
T-4
T-5
A T-7
T-8
T-9
0.00.20.40.60.81.01.21.41.61.8
AAAGGAATGG
5'- H3
turn
H2
“wing”
Poon (2012) Biochemistry 51, 4096-107
Reactivity to MnO4-
Possible explanationsIncreased stacking?
Major groove compression?
Helical twist controls base stacking and groove widths
Randall, Zechiedrich, and Pettitt. (2009) Nucleic Acids Res 37, 5568-77
OverwoundUnderwound
Sequence-dependent control of PU.1-DNA structures
• Sequence-specific PU.1-DNA complexes are grossly similar in structure (regardless of affinity)
• Nonspecific binding is a structurally distinguishable mode of low-affinity binding
• Hypersensitivity to DNase I is a hallmark of sequence-specific ETS-DNA binding ► deformability, rather than intrinsic curvature, is important for specific sequence recognition
• Flanking sequences appear to be overwound in low-affinity specific binding ► increased DNA bending?
• Specific ETS sites load additional ETS proteins at high concentrations in a negatively cooperative manner
PU.1 ETS-DNA interactions:Thermodynamics
“Thermodynamics is a wonderful structure with no content.”
Aharon Katchalsky
Sequence-specific thermodynamics
270 280 290 300 310 320 330
14
16
18
20
22
ln (K
B, M
-1)
Temperature, K
AGC/GTG
AAA/TGG
p SHBln ln 1
C TTKR T T
AGC/GTG
AGA/GTG
AAA/GTG
ACA/GTG
AAC/GTG
TGG/TGG
-60
-40
-20
0
20
40
60
kJ/m
ol a
t 298
K
H° TS°
p H
pS
( )
ln
H C T T
TS CT
Poon and Macgregor (2004) J Mol Biol, 335, 113-27Poon (2012) Biochemistry 51, 4096-107
-2.4 -2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8
10
12
14
16
18
20
22
0.091 0.11 0.14 0.17 0.20 0.25 0.30 0.37 0.45
[Na+], M
ln (K
B, M
-1)
ln ([Na+], M)-9 -8 -7 -6 -5 -4 -3 -2 -1
AAA/GTG
AGG/GTG
ACC/GTG
AGA/GTG
AGC/GTG
CAA/GTG
TAA/GTG
GAA/GTG
AGT/GTG
AAC/GTG
AAA/TGG
NS
SKobs
Bobs +
ln ψln[M ]KSK Z
Sequence-specific thermodynamics: electrostatics
AGC/GTG
AAA/TGG
NS
Poon and Macgregor (2004) J Mol Biol, 335, 113-27Poon (2012) J Biol Chem 287, 18297-307
cocrystalstructure
-153.5
-153.0
-152.5
-152.0
-151.5
-151.0
-150.5
-150.0
-149.5
-149.0
-148.5
Pow
er,
W
0 10 20 30 40 50
-50
-40
-30
-20
-10
0
10
20
0.0 0.4 0.8 1.2 1.6 2.0ETS:DNA
1/V
dq/
d[X
]t, k
J/m
ol
[ETS], M
0 10 20 30 40 50
0.0 0.2 0.5 0.7 0.9 1.2
[DNA], M
Calorimetric measurements revealcomplex ETS-DNA interactions
Empirical fit to asymmetric dimer
k1 = 1.2 ± 0.8 nMk2 = 88 ± 34 nMh1 = -0.25 ± 0.06 kJ/molh2 = -25.2 ± 0.8 kJ/mol
H1 = -22.8 ± 0.5 kJ/molH2 ~ +16 kJ/mol
5’-AGCGGAAGTG-3’
Poon (2012) Biochemistry 51, 4096-107Poon (2010) Anal Biochem, 400, 229-36
“Forward” titrationProtein DNA
“Reverse” titrationDNA protein
-155.5
-155.0
-154.5
-154.0
-153.5
-153.0
-152.5
-152.0
-151.5
Pow
er,
W
0 10 20 30 40 50-40
-30
-20
-10
0
10
20
0.0 0.8 1.7 2.5 3.3 4.1
1/V
dq/
d[X
]t, k
J/m
ol
[ETS], M
0 10 20 30 40 50
0.0 0.2 0.4 0.7 0.9 1.1ETS:DNA
[DNA], M
Empirical fit to asymmetric dimer
k1 = 1.8 ± 1.7 nMk2 = 54 ± 38 nMh1 = +5.1 ± 0.7 kJ/molh2 = -15.2 ± 0.9 kJ/mol
H1 = -13.6 ± 0.8 kJ/molH2 ~ +12 kJ/mol
High-affinity sequence(B motif)
5’-AAAGGAAGTG-3’
Poon (2012) Biochemistry 51, 4096-107
“Forward” titrationProtein DNA
“Reverse” titrationDNA protein
-154.5
-154.0
-153.5
-153.0
-152.5
-152.0
Pow
er,
W
0 10 20 30 40 50
-10
0
10
20
300.0 0.8 1.7 2.5 3.3 4.1ETS:DNA
1/V
dq/
d[X
]t, k
J/m
ol
[ETS], M
0 10 20 30 40 50
0.0 0.3 0.5 0.8 1.1 1.3
[DNA], M
Low-affinity sequence5’-AGCGGAATGG-3’
Empirical fit to asymmetric dimer
k1 = 10-7 Mk2 = 10-6 Mh1 = +12.0 ± 0.8 kJ/molh2 = -6.7 ± 1.0 kJ/mol
H1 = +4.2 ± 0.4 kJ/molH2 ~ +12 kJ/mol
1:2
Poon (2012) Biochemistry 51, 4096-107
“Forward” titrationProtein DNA
“Reverse” titrationDNA protein
ΔG°
1:1 complex
Absence of DNA DNA present
ETS
ETS ETS
.ETSETS 2:1 complex
ETS
Poon (2012) Biochemistry 51, 4096-107
Sequential binding of PU.1 ETS is thermodynamically distinct
5 10 15 20 25 30 35
-40
-20
0
20
40
60
HF
, kJ
/mol
Temperature, °C
5 10 15 20 25 30 35
0
10
20
30
40
150 mM Na+
250 mM Na+
HF
, kJ
/mol
Temperature, °C
Poon (2012) Biochemistry 51, 4096-107
First equivalent Second equivalent
Sequence-specific thermodynamics of PU.1 ETS-DNA interactions
• Thermodynamic profile of high-affinity PU.1-DNA binding Enthalpically-driven (ΔH° < 0) Entropic penalty (ΔS° < 0) Weak salt dependence (SKobs < N) (pH-insensitive between 6.5 to 9) Osmotically destabilized
• All sequence-specific PU.1-DNA binding appears to exhibit negative ΔCp
• DNA binding modulates dimerization of PU.1 ETS
Hydration controls the sequence specificity of PU.1
Water activity, osmolality, and osmotic stress
• Interactions that involve a change in hydration are sensitive to changes in water activity “Free” (= available) water
• Experimentally accessiblethrough osmolytes
• Also biologically relevant because the cell interior is highly crowded (up to 40 g/L) Substantial fraction of water is bound to macromolecules and
metabolites (= unavailable)
wOsm 55.5 lnm a
Water activity: a tasty approach to hydration
Source: AquaLab Decagon
+ Kobs
Preferential binding vs. preferential hydration
2
2 2
obs SH O S
H O H O
ln lnln lnd K d ad a d a
2H O 2 SA + B AB + H O + S Kobs
Kobs
Sensitivity to osmotic stress
2
2 2
obs SH O S
H O H O
ln lnln lnd K d ad a d a
2H O 2 SA + B AB + H O + S Kobs
N+O
O-HOOHO
OHO
HO
OH
HO
HO
OH
OH
OH
OO
OH
H
H
H
HH
HH
NH
N OH
I II III IV
Poon (2012) J Biol Chem 287, 18297-307
Poon (2012) J Biol Chem 287, 18297-307
●: ̶ osmolyte○: + osmolyte
PU.1-DNA complexes respond differentially to osmolytes
0 1 2 3 4 5 6
12
14
16
18
20
22
1.0 0.98 0.96 0.95 0.93 0.91 0.90
Water activity (aw)
ln (K
B, M
-1)
Osmolality
AGCGGAAGTG
AAAGGAATGG
AGCGAGAGTG
Poon (2012) J Biol Chem 287, 18297-307
Perturbation of PU.1-DNA complexes by osmolytes
○ TEG Betaine Sucrose▲ Nicotinamide
PWB B
w
ln ln Osm 55.5 ln 55.5K K
a
Poon (2012) J Biol Chem 287, 18297-307
G+A
5’-A
AAT
AA
GC
GG
AA
GTG
AA
AC
CA
AG
-3’
3’-GA
AC
CA
AA
GG
TAA
GG
AA
AATA
AA
-5’
High-affinitysequence
Low-affinitysequence
Poon (2012) J Biol Chem 287, 18297-307
DMS protection: GGAA strand
Poon (2012) J Biol Chem 287, 18297-307
337 Å3 / ~30 Å3 per H2O = ~11 H2O
Hydration in PU.1 ETS-DNA interactions
• Osmolytes discern 3 binding modes for PU.1-DNA binding: Net uptake ► high-affinity Net neutral ► low-affinity specific Net release ► nonspecific
• Lack of dependence on osmolyte identity for sequence-specific complexes implies a steric effect on water Core consensus is a water-sequestering cavity Accessible to bulk solvent in the low-affinity inaccessible in the high-affinity ETS-
DNA complex
• OS experiments predict a larger water uptake than structurally available Linked uptake of osmolyte? (Unlikely) ΔΓPW is an effective quantity, not necessarily a stoichiometry Immobilized waters form a highly cooperative hydration network
W
.ETSETS
W
Sequence-specific ETS recognition: a model
W WW W W
High-affinity binding Low-affinity binding
Extensive interfacial hydrationStrongly destabilized to osmotic stress
Water-sequestering core cavityProtection from DNase I, DMS, and ·OH
Enthalpically driven bindingImmobilize water network at entropic cost
Weak salt dependence
Small induced DNA curvatureLess overwound flanking segments
Poorly hydrated interfaceInsensitive to osmotic stress
“Leaky” core cavityProtection from DNase I and ·OH but not DMS
Entropically driven bindingIncreased backbone contacts (electrostatic)
Strong salt dependence
Pronounced induced DNA curvatureOverwound flanking segments
AGCGGAAGTG AAAGGAATGG
.ETSETS
W
Acknowledgements
Victor M. Bii
CollaboratorsDr. W. David Wilson (Chemistry,
Georgia State University)Dr. Arjan van der Vaart (Chemistry,
University of South Florida)
Financial supportAmerican Cancer Society (IRG -77-003-27)College of Pharmacy, WSU
Extra slides
Sequence-specific protein monomers, although not infrequent, have little to
contribute to DNA bending.
Dickerson and Chiu (1998) Biopolymers 44, 361-403
Quantitative footprinting: methodology
ETS
-bou
nd
A
Unb
ound
5'-AAATAAGCGGAAGTGAAACCAAG-3'
5'-AAATAAGCGGAAGTGAAACCAAG-3'5'-CTTGGTTTCACTTCCGCTTATTT-3'
5'-CTTGGTTTCACTTCCGCTTATTT-3'
0( )i i iQ a P x dx
Reactivity Q at position i is proportional to integrated intensity of corresponding peak centered at x = xi
2 20
1 γπ ( ) γiQ x x
1
n
ii
Q Q
Using a Lorentzian distribution to describe the line shape of Pi,
The trace is taken as a linear combination of individual band reactivities:
0 1000 2000 3000 4000 5000 6000
-155
-154
-153
-152
-151
-150
-149
-148
0 1000 2000 3000 4000 5000 6000
-155
-154
-153
-152
-151
-150
-149
-148
0 1000 2000 3000 4000 5000 6000
-155
-154
-153
-152
-151
-150
-149
-148
0 1000 2000 3000 4000 5000 6000
-155
-154
-153
-152
-151
-150
-149
-148
0 1000 2000 3000 4000 5000 6000
-155
-154
-153
-152
-151
-150
-149
-148
0 1000 2000 3000 4000 5000 6000
-155
-154
-153
-152
-151
-150
-149
-148
0 1000 2000 3000 4000 5000 6000
-155
-154
-153
-152
-151
-150
-149
-148
Nor
mal
titra
tion
(ETS
into
DN
A)
1:11:1
1:1
1:21:1
2:1
2:1
1:2 1:2
Rev
erse
titra
tion
(DN
A in
to E
TS)
ETS into buffer
AAAGGAATGGAAAGGAAGTG
Pow
er,
W
25°C, 150 mM Na+
time, s
AGCGGAAGTG
exothermic
Poon (2012) Biochemistry 51, 4096-107
ETS family of transcription factors
• Hematopoiesis• Organ cell fate• Neuronal development
Hollenhorst et al. (2011) Annu Rev Biochem 80, 437-471
YXETS
Sequence-specific control ofETS interactions: a model
NucleusExcess DNA, molecular confinement, crowding
ETS
ETSX
Cytosol
Binding site A Binding site B
X
H3
5’
5’
GGAA strand
TTCC strand
Dnase I hypersensitivity is stereospecific
5’-*
AA
ATA
AG
CG
GA
AG
TGA
AA
CC
AA
G-3
’ 3’-GA
AC
CA
AA
GG
TAA
GG
AA
AATA
AA
*-5’
C+T
3’
5’
-15 -11 -10 -9 -8 -7 -6 -5
500
1000
1500
2000
2500
3000
3500
4000
Inte
nsity
, AU
log [ETS, M]
-11 -10 -9 -8 -7 -6 -5A
T
A
A
G
C
G
G
A
A
G
T
G
A
A
A
A
T
A
A
G
C
G
G
A
A
G
T
G
A
A
A
log [ETS, M]
200.0
555.0
910.0
1265
1620
1975
2330
2685
3040
3395
3750
-11.0-10.9 -10 -9 -8 -7 -6 -5A
T
A
A
A
A
G
G
A
A
T
G
G
A
A
A
A
T
A
A
A
A
G
G
A
A
T
G
G
A
A
A
log [ETS, M]
200.0
450.0
700.0
950.0
1200
1450
1700
1950
2200
2450
2700
-15 -11 -10 -9 -8 -7 -6 -5
500
1000
1500
2000
2500
3000
Inte
nsity
, AU
log [ETS, M]
High-affinity sequence
Low-affinity sequence
DNase I protection: GGAA strand-log [ETS, M]55
10 10
Reciprocal ETS-DNA equilibria: a scheme
“Forward” titrationProtein DNA
“Reverse” titrationDNA protein
DNA binding is negatively cooperative withrespect to protein dimerization
ETS dimerization is negatively cooperative withrespect to DNA binding
P: PU.1 ETSD: DNA
Poon (2012) Biochemistry 51, 4096-107
Unbound PU.1 ETS is a weak dimer
0 2 4 6 8 10 12 14 16 18
0.026
0.028
0.030
0.032
0.034
0.036
(K C
)/R,
kDa-1
C, g mL-1
Apparent MW: 27.5 ± 1.1 kDaFormula weight: 13.0 kDaApparent stoichiometry: 2.1 ± 0.1(Concentration: 0.9 mM)
SLS DLS
24.4 kDa
Poon (2012) Biochemistry 51, 4096-107
Unbound PU.1 ETS is a weak dimer
0 5 10 15 20 25
18.2
17.7
18 µM
Elution volume, mL
130 µM
-6.0 -5.5 -5.0 -4.5 -4.0 -3.5
17.4
17.6
17.8
18.0
18.2
Elu
tion
volu
me,
mL
log (PU.1 ETS, M)
Poon (2012) Biochemistry 51, 4096-107 and unpublished results
KD ~ 20 µM
SEC
ETS-DNA interactions: summary
• The PU.1 ETS domain assumes different oligomeric states in the unbound vs. DNA-bound states.
• These oligomeric states are thermodynamically distinguishable, implying possible distinct conformations between the free and bound states.
• Conformational changes induced by DNA binding may represent specific permissive or inhibitory states for protein-protein interactions.
-6 -4 -2 0 2 4 6-6
-4
-2
0
2
4
6
8
AGC
ACC
AGG
TAC
AGT
ACT ACT
TGA
G
° fro
m A
AA
G°int
from AAA
AAA AXA
AAY AXY
ΔΔG° (AXA)
ΔΔ
G°
(AAY
) ΔΔG° (AXY)
Cooperative coupling by flanking bases
AAAGGAAGTG
int AXY AXA AAY AAA( )G G G G G
Poon and Macgregor (2003) J Mol Biol, 328, 805-19