Binding Studies on Trafficking Proteins
Using Microcalorimetry
McMahon lab
Neurobiology Division
Laboratory of Molecular Biology
Cambridge
Clathrin Mediated Endocytosis
Binding
Recruitment
Coating
Receptor
Ligand
AP adaptor complex
Regulatory adaptor
Clathrin
Budding
Uncoating
Dynamin
Receptor Mediated Endocytosis
a
b
c
a) Yolk protein (Gilbert und Perry 1979)
b) Low Density Lipoprotein (Anderson et al. 1977)
c) Virus particle (Matlin et al. 1981)
Replica of the inner membrane surface
(Heuser and Anderson 1989)
AP Adaptor Complex
, , , 
1-4
Appendage
binds regulators
Hinge
binds clathrin
Trunk
1-4
1-4
binds lipids and
membrane proteins
Collins et al. 2002
AP-1 ():
AP-2 ():
AP-3 ():
AP-4 ():
TGN / Endosome
Plasma membrane
Lysosome
TGN
AP Trafficking Pathways
Plasma membrane
AP-2
Endosome
Lysosome
AP-4
AP-3
GGA
AP-1
Lysosome-related
Organelle
AP-3
Trans-Golgi-Network
AP Appendage Domains
DP(F/W)
FxDxF
DP(F/W)
DPW
FxxF
-Adaptin
Owen et al. 1999
Brett et al. 2002
-Adaptin
Owen et al. 2000
-Adaptin
Kent et al. 2002
Nogi et al. 2002
Regulatory Adaptors
Epsin1
EpsinR
AP180
Dab2
Eps15
Amphiphysin1
Aminoacid
0
250
500
750
1000
DOMAIN
PARTNER
SH3
PTB
ANTH/ENTH
BAR
EH
Clathin-Box
DxF/W
NPF
PxxPxR
PxxPxR
Receptor and Lipids
Lipids
Lipids
NPF
Clathrin
- and -Adaptin
EH
SH3
Interactions in Trafficking
Amphiphysin
Receptor
Lipids
LLDLD
Yxx
or LL
AP-Complex
DxF or
FxxF
Eps15
Clathrin
LLDLD
NPF
Epsin1
EpsinR
AP180
Dab2
Determination of Binding Constants
Definition of Association and Dissociation Constants:
k1
For a binding reaction at equilibrium: P + L
PL
k-1
[P]free = conc. of free protein
[L]free = conc. of free ligand
[PL] = conc. of PA complex
k1 = rate constant for formation of [PL]
k-1 = rate constant for breakdown of [PL]
The rate of formation of [PL] is k1 [P]free [L]free, where k1 is a second order rate constant with units of l/mol-1s-1.
The rate of breakdown of [PL] is k-1 [PL], where k-1 is a first order rate constant with units of s-1.
At equilibrium, the rate of formation of [PL] equals the rate of its breakdown, so k1 [P]free[L]free= k-1 [PL].
Also recall that:
KD = k-1 / k1 = [P]free [L]free/ [PL] = 1 / KA
KD is given in units of concentration (e.g., mol/l)
Or, in terms of fraction of protein binding sites occupied (y), which is often convenient to measure:
y = [PL] / ([P]free+ [PL])
= [L]free / ([L]free + KD)
• Use [PL] = KA [P]free [L]free
• Divide through by KA
• Replace KA by 1 / KD
Determination of Binding Constants
Special cases:
y = [L]free / ([L]free + KD)
For [L]free = 0:
y =0
nothing bound
For [L]free :
y =1
full occupancy
For [L]free = KD:
y = 0.5
half occupancy
Two possible ways to determine binding constants:
1.
Measure bound and free ligand at equilibrium as a function of concentration
2.
Measure association and dissociation rate constants and use these to calculate binding constants
Methods to determine Binding Constants
Spectroscopy
(Fluorescence, UV/Vis, CD)
Signal
change of absorption
or emission of light
Information
KD (10-4-10-11M)
Advantage
in solution
Disadvantage
probe needed
Microcalorimetry
heat of binding
KD (10-3-10-11M)
H, S, n
no labels,
in solution
direct access to H
direct access to n
large sample
Surface Plasmon Resonance
change of refractive
index due to mass
KD (10-3-10-13M)
k1, k-1
small sample,
automated
surface coupled,
ligand must have
large mass
Stopped-Flow
coupled to spectroscopy KD (10-3-10-12M)
k1, k-1
fast
probe needed
Analytical Ultracentrifugation absorption at different
radii for different times
KD (10-3-10-8M)
good for
homomeric
interactions
slow
Nuclear Magnetic Resonance shift of magnetic
resonance frequency
KD (10-3-10-6M)
in solution,
structural
information
slow,
large sample,
expensive
can be most
sensitive
sometimes
inaccurate
Binding Assays
various, e.g. SDS-PAGE, KD (10-3-10-15M)
densitometry, radioactivity
Isothermal Titration Calorimetry (ITC)
Isothermal Titration Calorimetry (ITC)
Taken from Micro Cal website
Isothermal Titration Calorimetry (ITC)
Review of Free Energies, Enthalpies, and Entropies of Binding
G°bind = RT lnKD (where R= 1.98 cal mol–1 K-1; T= 273.2 K, and RT =0.62 kcal/mol at 37°C)
Note log relationship between free energy and binding constants
Recall that G°bind is relative to standard conditions (typically 1M reactants, 25 °C, standard salt)
A convenient rule of thumb is that a 10-fold change in binding constant corresponds to 1.4 kcal / mol.
G°A1-A2 = RT ln(KDA1 / KDA2)= (0.62 kcal / mol)ln(10-8 M / 10-7M) = -1.4 kcal / mol
How many kcal / mol change in free energy do you need to change KD 100-fold?
Isothermal Titration Calorimetry (ITC)
Review of Free Energies, Enthalpies, and Entropies of Binding
G°bind = RT lnKD (where R= 1.98 cal mol–1 K-1; T= 273.2 K, and RT =0.62 kcal/mol at 37°C)
Note log relationship between free energy and binding constants
Recall that G°bind is relative to standard conditions (typically 1M reactants, 25 °C, standard salt)
A convenient rule of thumb is that a 10-fold change in binding constant corresponds to 1.4 kcal / mol.
G°A1-A2 = RT ln(KDA1 / KDA2)= (0.62 kcal / mol)ln(10-8 M / 10-7M) = -1.4 kcal / mol
How many kcal / mol change in free energy do you need to change KD 100-fold?
 - 2.8 kcal / mol
Isothermal Titration Calorimetry (ITC)
Review of Free Energies, Enthalpies, and Entropies of Binding
G°bind = RT lnKD (where R= 1.98 cal mol–1 K-1; T= 273.2 K, and RT =0.62 kcal/mol at 37°C)
Note log relationship between free energy and binding constants
Recall that G°bind is relative to standard conditions (typically 1M reactants, 25 °C, standard salt)
A convenient rule of thumb is that a 10-fold change in binding constant corresponds to 1.4 kcal / mol.
G°A1-A2 = RT ln(KDA1 / KDA2)= (0.62 kcal / mol)ln(10-8 M / 10-7M) = -1.4 kcal / mol
How many kcal / mol change in free energy do you need to change KD 100-fold?
 - 2.8 kcal / mol
Recall also that free energy has enthalpy and entropy components:
G° = H° -T S° (and therefore) –RTlnKA= H° -T S°
When is an interaction strong?
G° must be large and negative
 H° must be large and negative (gain new bonds)
 S° must be large and positive (gain more entropy)
Isothermal Titration Calorimetry (ITC)
Time (min)
90
60
30
0
kcal/mol Ligand
cal/s
0
-2
-4
0
affinity: 1/Kd
-5
enthalpy: H
-10
stochiometry: N
-15
0.0
0.5
1.0
1.5
Ligand / Protein
2.0
Isothermal Titration Calorimetry (ITC)
Time (min)
90
60
30
0
kcal/mol Ligand
cal/s
0
-2
-4
0
affinity: 1/Kd
-5
enthalpy: H
-10
stochiometry: N
-15
0.0
0.5
1.0
1.5
Ligand / Protein
2.0
G  14.1
H  19.9
TS  5.8
Binding Specificity
Amph1 1-372
DNF-ANF
DNF-DPF
DNF-RPF
DNF-DPP
DNF-DPW
DNF-DGF
DNF-DIF
DNF-DLF
DNF-DAF
DNF-DDF
DNF-DSF
DNF-EPL
Extract
Extract
DNF+DPF-SGA
DPF-SGA
DNF-SGA
Amph1 1-372
-Adaptin and Amphiphysin
-Adaptin
-Adaptin
-Adaptin
-Adaptin
Sequence
rAmphiphysin1
rAmphiphysin2
INFFEDNFVPEINVTTPSQNEVLEVKKEE TLLDLDFDPFKPDVTPAGSAAATHSPMSQTLPWDLW
LSLFDDAFVPEISVTTPSQFEAPGPFSEQASLLDLDFEPLPPVASPVKAPTPSG
QSIPWDLW
Praefcke et al. 2004
Olesen et al. 2007
Binding Specificity
-Adaptin and Amphiphysin
Time (min)
kcal/mol Peptide cal/s
0
30
60
90
5
7
DxF Peptide
0
8
-5
12
0
-2
-4
-6
-8
7
8
12
0.0
0.5
1.0
1.5
2.0
2.5
Sequence
KD (M)
DNF 7mer
DNF to RNF 7mer
DNF 8mer
DNF 12mer
DNF to DPF 12mer
DNF to DAF 12mer
DNF FE-change
FEDNFVP
FERNFVP
FEDNFVPE
INFFEDNFVPEI
INFFEDPFVPEI
INFFEDAFVPEI
INFEFDNFVPEI
21
no binding
28
2.5
120
21
180
DPF 12mer
DPF to DNF-12mer
LDLDFDPFKPDV
LDLDFDNFKPDV
190
no binding
3.0
DNF-Peptide / -Appendage
Sequence
rAmphiphysin1
rAmphiphysin2
INFFEDNFVPEINVTTPSQNEVLEVKKEE TLLDLDFDPFKPDVTPAGSAAATHSPMSQTLPWDLW
LSLFDDAFVPEISVTTPSQFEAPGPFSEQASLLDLDFEPLPPVASPVKAPTPSG
QSIPWDLW
Praefcke et al. 2004
Olesen et al. 2007
Binding Specificity
-Adaptin and Amphiphysin
Time (min)
kcal/mol Peptide cal/s
0
30
60
90
5
7
DxF Peptide
0
8
-5
12
0
-2
-4
-6
-8
7
8
12
0.0
0.5
1.0
1.5
2.0
2.5
Sequence
KD (M)
DNF 7mer
DNF to RNF 7mer
DNF 8mer
DNF 12mer
DNF to DPF 12mer
DNF to DAF 12mer
DNF FE-change
FEDNFVP
FERNFVP
FEDNFVPE
INFFEDNFVPEI
INFFEDPFVPEI
INFFEDAFVPEI
INFEFDNFVPEI
21
no binding
28
2.5
120
21
180
DPF 12mer
DPF to DNF-12mer
LDLDFDPFKPDV
LDLDFDNFKPDV
190
no binding
Synaptojanin
HIP1
Dab2
LDGFEDNFDLQS
DNKFDDIFGSSF
QSNFLDLFKGNA
3.0
DNF-Peptide / -Appendage
4.5
100
no binding
DNF-site is 80 fold stronger than DPF-site
Very good correlation between Western Blots and ITC
Residue at position 4 in FxDxF is important (N>S>A>I>P>L)
Prediction for other proteins possible
Praefcke et al. 2004
Olesen et al. 2007
Lipid Binding
Epsin1 ENTH domain
P
S
P
S
P S
P
S
P
S
P
S
PtdCho
Blank
PtdEth
PtdSer
PtdIns(5)P
PtdAcid
PtdIns(4)P
PtdIns(3,4,5)P3
PtdIns(3)P
PtdIns(3,5)P2
PtdIns
PtdIns(4,5)P2
LysoPtdCho
PtdIns(3,4)P2
LysoPtdAcid
Sphing-1-P
P S
Ford et al. 2002
Lipid Binding
Epsin1 ENTH domain
Time (min)
0
30
60
90
120
150
180
kcal/mol InsPx
cal/s
0
KD (M)
-5
 Ins(1,4)P2
>1,000
-10
 Ins(1,5)P2
>1,000
-15
 Ins(1,3,5)P3
120
0
 Ins(1,4,5)P3
3.6
Ins(1,3,4,5)P4
4.1
InsP6
0.55
-10
diC8PtdIns(4,5)P2
-20
0.0
0.5
1.0
1.5
2.0
0.85
2.5
InsPx / Epsin1 ENTH
Good correlation between ITC and other binding assays
Head groups are a good model for the lipid molecules
Ford et al. 2002
Lipid Binding
Epsin1 ENTH domain
Time (min)
0
30
60
90
120
150
cal/s
1.0
Liposomes
0.5
Liposomes
+ ENTH
0.0
kcal/mol Protein
-0.5
0
-10
Epsin1
0.0
0.5
Disabled2
1.0
1.5
2.0
2.5
3.0
3.5
Protein / PI(4,5)P2 in outer leaflet
Data for Epsin1-ENTH with liposomes is different from control protein
 ITC reveals tubulation of liposomes by the ENTH domain
Ford et al. 2002
Multiple Binding Sites
EpsinR and -Adaptin
D422R
E391R
D371R
D349R
D328R
Point Mutations
D325R
291-334
291-345
291-379
291-397
Clathrin
291-625
-Adaptin
<349
<345
<334
<328
<325
291-426
<422
<397
<391
<379
(291)AHYTGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS
<371
291-429
Truncations
FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426)
Mills et al. 2003
Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
0
30
60
90
120
150
180
210
cal/s
0
-2
kcal/mol EpsinR
-4
0
-10
-20
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
EpsinR 291-426 / -Adaptin-Appendage
One Site Model
N
0.61
KD (M)
3.8
Two Site Model
N1
KD (M)
1.2
0.26
N2
KD (M)
2.4
9.3
Mills et al. 2003
Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
Time (min)
0
30
60
90
120
150
180
0
210
60
90
120
150
0
cal/s
0
cal/s
30
-2
-2
-4
kcal/mol EpsinR
0
-10
-20
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
EpsinR 291-426 / -Adaptin-Appendage
kcal/mol -Adaptin
-6
-4
0
-5
-10
0.0
KD (M)
3.8
Two Site Model
N1
KD (M)
1.2
0.26
N2
KD (M)
2.4
9.3
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
-Adaptin-Appendage / EpsinR 291-426
One Site Model
N
0.61
0.5
swap cell and syringe content
One Site Model
N
KD (M)
1.3
19
Two Site Model
N1
KD (M)
0.90
0.72
N2
KD (M)
0.84
51
Mills et al. 2003
Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
0
kcal/mol Peptide cal/s
5
0
30
60
90
120
Peptide
P3
KD (M)
-Adaptin
no binding
230
110
> 220
48
EpsinR
P1-SGDLVDLFDGTS
P2-TGGSADLFGGFA
P3-SADLFGGFADFG
P4-FGGFADFGSAAA
P5-TSGNGDFGDWSA
P5
-5
0
-2
-4
-6
P3
P5
0.0
0.5
1.0
1.5
EpsinR Peptide / Adaptin-Appendage
291(AHY)TGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS
P1
P2
P3
P4
FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426)
P5
Mills et al. 2003
Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
0
30
60
90
120
P3
P5
Sy
-10
0
-2
-4
P3
-6
P5
Sy
0.0
0.5
1.0
1.5
2.0
2.5
KD (M)
EpsinR
P1-SGDLVDLFDGTS
P2-TGGSADLFGGFA
P3-SADLFGGFADFG
P4-FGGFADFGSAAA
P5-TSGNGDFGDWSA
-Adaptin
no binding
230
110
> 220
48
-Synergin
PEEDDFQDFQDA
Eps15
SFGDGFADFSTL
Epsin1
EPDEFSDFDRLR
EF-hand
NEDDFGDFGDFG
13
180
200
8
<349
EpsinR Peptide / Adaptin-Appendage
Peptide
291(AHY)TGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS
P3
<371
kcal/mol Peptide cal/s
0
FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426)
P5
Mills et al. 2003
Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
kcal/mol Peptide cal/s
0
0
30
60
90
120
P3
P5
Sy
-10
0
-2
-4
P3
-6
P5
Sy
0.0
0.5
1.0
1.5
2.0
2.5
EpsinR Peptide / Adaptin-Appendage
Peptide
KD (M)
EpsinR
P1-SGDLVDLFDGTS
P2-TGGSADLFGGFA
P3-SADLFGGFADFG
P4-FGGFADFGSAAA
P5-TSGNGDFGDWSA
-Adaptin
no binding
230
110
> 220
48
-Synergin
PEEDDFQDFQDA
Eps15
SFGDGFADFSTL
Epsin1
EPDEFSDFDRLR
EF-hand
NEDDFGDFGDFG
EpsinR contains two binding sites for -Adaptin
Identification of consensus motif using peptides
Motif is also present in other trafficking proteins
13
180
200
8
Isothermal Calorimet ry T itration with EpsinR N3 const ruct
N3 + -appendage Average
N3 D342R + -appendage Average
N3 D349R + -appendage Average
N3 D371R + -appendage Average
N1
N2
0.91
0.95
0.91
0.87
0.91
1.0
0.93
0.78
N3 + -appendage Average
1.0
58
-18390
-12870
N3 D342R + -appendage
0.56
54
-33400
-27860
-appendage + N3
1.07
GST-GGA1 + N3
0.72
KD (M)
H (cal mol-1)
TS (cal mol-1)
1.48
KD1
0.68
.85
3.2
4.9
0.78
95
KD2
45
160
54
68
22
² H1
² H2
T² S1
T² S2
-12780
-13100
-11250
-11630
-10400
-8970
-11370
-11490
-4790
-5230
-4120
-4750
-4780
-4070
-5820
-6090
-18530
-4310
-11070
Exothermic
-10595
{ +1730 }
-5860
Decrease
in Entropy
Except in{..}
Mills et al. 2003
Temperature Dependence
Synaptotagmin C2A domain and Calcium
Time (min)
0
30
60
90
120
150
cal/s
10
180
10 °C
N
KD (M)
H (cal/mol)
5
25 °C
0
kcal/mol Ca2+
-5
2
1
0
0
2
4
6
8
Ca2+ / Synaptotagmin C2A
Two calcium binding sites per C2A domain
No robust fit for two site model
10°C
1.8
450
+3080
25°C
2.1
340
+1830
Temperature Dependence
Synaptotagmin C2A domain and Calcium
Time (min)
0
30
60
90
120
150
kcal/mol Ca2+
cal/s
10
180
10°C
N1
1.8
KD1 (M)
450
H1 (cal/mol) +3080
10 °C
25 °C
0
37 °C
25°C
2.1
340
+1830
37°C
0.9
103
-530
2
N2
KD2 (M)
H2 (cal/mol)
1
0
0
2
4
6
8
Ca2+ / Synaptotagmin C2A
At higher temperature the reaction is more exothermic
At 37°C the two sites can be fitted and resolved
0.9
410
+3770
Summary
Microcalorimetry
• is a versatile technique to study biological interactions in solution
• is applicable to ligands such as proteins, peptides, lipids, liposomes, DNA, ions,…
• gives direct access to all thermodynamic parameters from one single experiment
• allows for the precise determination of stochiometry of binding reactions