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Binding Studies on Trafficking ProteinsUsing Microcalorimetry
McMahon lab
Neurobiology Division Laboratory of Molecular Biology
Cambridge
Clathrin Mediated Endocytosis
Binding Coating Recruitment
Uncoating
Receptor
Ligand
AP adaptor complex
Regulatory adaptor
Clathrin
DynaminBudding
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)
a
c
b
Receptor Mediated Endocytosis
AP Adaptor Complex
AP-1 (): TGN / EndosomeAP-2 (): Plasma membrane AP-3 (): LysosomeAP-4 (): TGN
1-4 1-4
, , , 1-4
Appendagebinds regulators
Hingebinds clathrin
Trunkbinds lipids andmembrane proteins
Collins et al. 2002
AP Trafficking Pathways
AP-2
AP-3
AP-1AP-4 AP-3GGA
Plasma membrane
Trans-Golgi-Network
Lysosome
Endosome
Lysosome-relatedOrganelle
AP Appendage Domains
Owen et al. 2000 Owen et al. 1999 Brett et al. 2002
FxDxFDP(F/W)
DPW
FxxF
-Adaptin -Adaptin -AdaptinKent et al. 2002 Nogi et al. 2002
DP(F/W)
Regulatory Adaptors
Aminoacid250 1000500 7500
Dab2
AP180
EpsinR
Eps15
Amphiphysin1
Epsin1
DOMAIN PARTNERSH3 PxxPxRPTB Receptor and LipidsANTH/ENTH LipidsBAR LipidsEH NPFClathin-Box ClathrinDxF/W - and -AdaptinNPF EHPxxPxR SH3
Amphiphysin
Eps15 Epsin1EpsinRAP180Dab2
FEDNF
DxF or FxxFLLDLD
LL
DL
D
LLDLD
NPF
DxF orFxxF
Yxxor LL
Interactions in Trafficking
Clathrin
Receptor Lipids
AP-Complex
Definition of Association and Dissociation Constants:
k1 [P]free = conc. of free proteinFor a binding reaction at equilibrium: P + L PL [L]free = conc. of free ligand k-1 [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]) • Use [PL] = KA [P]free [L]free
• Divide through by KA
• Replace KA by 1 / KD
= [L]free / ([L]free + KD)
Determination of Binding Constants
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:
• Measure bound and free ligand at equilibrium as a function of concentration
• Measure association and dissociation rate constants and use these to calculate binding constants
Methods to determine Binding Constants
Signal Information Advantage DisadvantageSpectroscopy change of absorption KD (10-4-10-11M) in solution probe needed(Fluorescence, UV/Vis, CD) or emission of light
Microcalorimetry heat of binding KD (10-3-10-11M) no labels, large sampleH, S, n in solution
direct access to Hdirect access to n
Surface Plasmon Resonance change of refractive KD (10-3-10-13M) small sample, surface coupled,index due to mass k1, k-1 automated ligand must have
large mass
Stopped-Flow coupled to spectroscopy KD (10-3-10-12M) fast probe neededk1, k-1
Analytical Ultracentrifugation absorption at different KD (10-3-10-8M) good for slowradii for different times homomeric
interactions
Nuclear Magnetic Resonance shift of magnetic KD (10-3-10-6M) in solution, slow,resonance frequency structural large sample,
information expensive
Binding Assays various, e.g. SDS-PAGE, KD (10-3-10-15M) can be most sometimesdensitometry, radio- sensitive inaccurateactivity
Isothermal Titration Calorimetry (ITC)
Isothermal Titration Calorimetry (ITC)
Taken from Micro Cal website
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)
Isothermal Titration Calorimetry (ITC)
-4
-2
0
0 30 60 90
0.0 0.5 1.0 1.5 2.0
-15
-10
-5
0
Ligand / Protein
cal
/skc
al/m
ol L
igan
d
Time (min)
stochiometry: N
enthalpy: H
affinity: 1/Kd
Isothermal Titration Calorimetry (ITC)
-4
-2
0
0 30 60 90
0.0 0.5 1.0 1.5 2.0
-15
-10
-5
0
Ligand / Protein
cal
/skc
al/m
ol L
igan
d
Time (min)
stochiometry: N
enthalpy: H
affinity: 1/Kd
€
G = −14.1
ΔH = −19.9
TΔS = −5.8
Am
ph
1 1-
372
DN
F-S
GA
DP
F-S
GA
DN
F+
DP
F-S
GA
Ext
rac
t
-Adaptin
-Adaptin
SequencerAmphiphysin1 INFFEDNFVPEINVTTPSQNEVLEVKKEE TLLDLDFDPFKPDVTPAGSAAATHSPMSQTLPWDLW rAmphiphysin2 LSLFDDAFVPEISVTTPSQFEAPGPFSEQASLLDLDFEPLPPVASPVKAPTPSG QSIPWDLW
-Adaptin
-Adaptin
Am
ph
1 1-
372
DN
F-A
NF
DN
F-D
PF
DN
F-R
PF
DN
F-D
PP
DN
F-D
PW
DN
F-D
GF
DN
F-D
IFD
NF
-DL
FD
NF
-DA
FD
NF
-DD
FD
NF
-DS
FD
NF
-EP
LE
xtra
ct
Binding Specificity-Adaptin and Amphiphysin
Praefcke et al. 2004
Olesen et al. 2007
-5
0
5
0 30 60 90
0.0 0.5 1.0 1.5 2.0 2.5 3.0
-8
-6
-4
-2
0
DNF-Peptide / -Appendage
Time (min)
cal
/skc
al/
mo
l P
ep
tid
e
12
7
8
8
12
7
SequencerAmphiphysin1 INFFEDNFVPEINVTTPSQNEVLEVKKEE TLLDLDFDPFKPDVTPAGSAAATHSPMSQTLPWDLW rAmphiphysin2 LSLFDDAFVPEISVTTPSQFEAPGPFSEQASLLDLDFEPLPPVASPVKAPTPSG QSIPWDLW
DxF Peptide Sequence KD (M)
DNF 7mer FEDNFVP 21DNF to RNF 7mer FERNFVP no bindingDNF 8mer FEDNFVPE 28DNF 12mer INFFEDNFVPEI 2.5DNF to DPF 12mer INFFEDPFVPEI 120DNF to DAF 12mer INFFEDAFVPEI 21DNF FE-change INFEFDNFVPEI 180
DPF 12mer LDLDFDPFKPDV 190DPF to DNF-12mer LDLDFDNFKPDV no binding
Binding Specificity-Adaptin and Amphiphysin
Praefcke et al. 2004
Olesen et al. 2007
-5
0
5
0 30 60 90
0.0 0.5 1.0 1.5 2.0 2.5 3.0
-8
-6
-4
-2
0
DNF-Peptide / -Appendage
Time (min)
cal
/skc
al/
mo
l P
ep
tid
e
12
7
8
8
12
7 DxF Peptide Sequence KD (M)
DNF 7mer FEDNFVP 21DNF to RNF 7mer FERNFVP no bindingDNF 8mer FEDNFVPE 28DNF 12mer INFFEDNFVPEI 2.5DNF to DPF 12mer INFFEDPFVPEI 120DNF to DAF 12mer INFFEDAFVPEI 21DNF FE-change INFEFDNFVPEI 180
DPF 12mer LDLDFDPFKPDV 190DPF to DNF-12mer LDLDFDNFKPDV no binding
Synaptojanin LDGFEDNFDLQS 4.5HIP1 DNKFDDIFGSSF 100Dab2 QSNFLDLFKGNA no binding
Binding Specificity-Adaptin and Amphiphysin
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 BindingEpsin1 ENTH domain
PtdCho
PtdEth
PtdIns(5)P
PtdIns(4)P
PtdIns(3)P
PtdIns
LysoPtdCho
LysoPtdAcid
Blank
PtdSer
PtdAcid
PtdIns(3,4,5)P3
PtdIns(3,5)P2
PtdIns(4,5)P2
PtdIns(3,4)P2
Sphing-1-P
No
Lipo
som
es
Ptd
Ins(
3,4,
5)P 3
P S P S P S P S P S P S P S
Ptd
Ins(
3)P
Ptd
Ins(
4)P
Ptd
Ins(
3,4)
P 2
Ptd
Ins(
4,5)
P 2
Ptd
Ins(
3,5)
P 2
Ford et al. 2002
Lipid Binding
-15
-10
-5
0
0 30 60 90 120 150 180
0.0 0.5 1.0 1.5 2.0 2.5
-20
-10
0
KD (M)
Ins(1,4)P2 >1,000
Ins(1,5)P2 >1,000
Ins(1,3,5)P3 120
Ins(1,4,5)P3 3.6
Ins(1,3,4,5)P4 4.1
InsP6 0.55
diC8PtdIns(4,5)P2 0.85
Time (min)
InsPx / Epsin1 ENTH
kca
l/m
ol
Ins
Px
cal
/s
Lipid BindingEpsin1 ENTH domain
Good correlation between ITC and other binding assays
Head groups are a good model for the lipid molecules
Ford et al. 2002
-0.5
0.0
0.5
1.00 30 60 90 120 150
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
-10
0
Lipid BindingTime (min)
Protein / PI(4,5)P2 in outer leaflet
kca
l/m
ol
Pro
tein
cal
/s
Disabled2Epsin1
Liposomes Liposomes+ ENTH
Lipid BindingEpsin1 ENTH domain
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 SitesEpsinR and -Adaptin
291-625Clathrin
-Adaptin
291
-429
291
-397
291
-379
291
-345
291
-334
D32
5R
D32
8R
D34
9R
D37
1R
E39
1R
D42
2R
Truncations Point Mutations
291-426
(291)AHYTGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS
FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426)
<3
25
<3
28
<3
49
<3
71
<3
91
<4
22
<3
97
<3
79
<3
34
<3
45
Mills et al. 2003
-4
-2
0
0 30 60 90 120 150 180 210
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
-20
-10
0
Multiple Binding Sites
EpsinR 291-426 / -Adaptin-Appendage
Time (min)
kca
l/m
ol
Ep
sin
Rc
al/s
One Site ModelN KD (M)0.61 3.8
Two Site ModelN1 KD (M)1.2 0.26N2 KD (M)2.4 9.3
Multiple Binding SitesEpsinR and -Adaptin
Mills et al. 2003
-4
-2
0
0 30 60 90 120 150 180 210
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
-20
-10
0
Multiple Binding Sites
EpsinR 291-426 / -Adaptin-Appendage
Time (min)
kca
l/m
ol
Ep
sin
Rc
al/s
-6
-4
-2
0
0 30 60 90 120 150
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
-10
-5
0
Time (min)
-Adaptin-Appendage / EpsinR 291-426
cal
/skc
al/
mo
l -
Ad
apti
n
One Site ModelN KD (M)0.61 3.8
Two Site ModelN1 KD (M)1.2 0.26N2 KD (M)2.4 9.3
One Site ModelN KD (M)1.3 19
Two Site ModelN1 KD (M)0.90 0.72N2 KD (M)0.84 51
swap cell and syringe content
Multiple Binding SitesEpsinR and -Adaptin
Mills et al. 2003
Multiple Binding Sites
Peptide KD (M) EpsinR -Adaptin P1-SGDLVDLFDGTS no bindingP2-TGGSADLFGGFA 230P3-SADLFGGFADFG 110P4-FGGFADFGSAAA > 220P5-TSGNGDFGDWSA 48
-5
0
5
0 30 60 90 120
0.0 0.5 1.0 1.5
-6
-4
-2
0
P5
P5
EpsinR Peptide / Adaptin-Appendage
cal
/skc
al/
mo
l P
ep
tid
e
P3
P3
P1P3
P4
P2
P5
291(AHY)TGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS
FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426)
Time (min)
Multiple Binding SitesEpsinR and -Adaptin
Mills et al. 2003
-10
0
0 30 60 90 120
0.0 0.5 1.0 1.5 2.0 2.5
-6
-4
-2
0
Multiple Binding Sites
Peptide KD (M) EpsinR -Adaptin P1-SGDLVDLFDGTS no bindingP2-TGGSADLFGGFA 230P3-SADLFGGFADFG 110P4-FGGFADFGSAAA > 220P5-TSGNGDFGDWSA 48
-SynerginPEEDDFQDFQDA 13Eps15SFGDGFADFSTL 180Epsin1EPDEFSDFDRLR 200EF-handNEDDFGDFGDFG 8
P3
P5
P5
P5
EpsinR Peptide / Adaptin-Appendage
cal
/skc
al/
mo
l P
ep
tid
e
P3
P3
<3
71
<3
49
291(AHY)TGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS
FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426)
Time (min)
Sy
Sy
Multiple Binding SitesEpsinR and -Adaptin
Mills et al. 2003
-10
0
0 30 60 90 120
0.0 0.5 1.0 1.5 2.0 2.5
-6
-4
-2
0
Multiple Binding Sites
Peptide KD (M) EpsinR -Adaptin P1-SGDLVDLFDGTS no bindingP2-TGGSADLFGGFA 230P3-SADLFGGFADFG 110P4-FGGFADFGSAAA > 220P5-TSGNGDFGDWSA 48
-SynerginPEEDDFQDFQDA 13Eps15SFGDGFADFSTL 180Epsin1EPDEFSDFDRLR 200EF-handNEDDFGDFGDFG 8
P5
P5
EpsinR Peptide / Adaptin-Appendage
cal
/skc
al/
mo
l P
ep
tid
e
P3
P3
Time (min)
Sy
Sy
Multiple Binding SitesEpsinR and -Adaptin
EpsinR contains two binding sites for -Adaptin
Identification of consensus motif using peptides
Motif is also present in other trafficking proteins
Isothermal Calorimetry Titration with EpsinR N3 constructN1 N2 KD1 KD2 ∆H1 ∆H2 T∆S1 T∆S2
N3 + -appendag e Average 0.91 0.91 0.68 45 -12780 -10400 -4790 -4780N3 D342 R + -appendag e Average 0.95 1.0 .85 160 -13100 -8970 -5230 -4070N3 D349 R + -appendag e Average 0.91 0.93 3.2 54 -11250 -11370 -4120 -5820N3 D371 R + -appendag e Average 0.87 0.78 4.9 68 -11630 -11490 -4750 -6090
N3 + -appendag e Average 1.0 58 -18390 -12870
N3 D342 R + -appendage 0.56 54 -33400 -27860
-appendag + 3e N 1.07 1.48 0.78 22 -18530 -4310 -10595 +1730
GST-GGA1 + 3N 0.72 95 -11070 -5860
KD (M)H (cal mol-1)TS (ca l mol-1)
Exothermic Decreasein EntropyExcept in{..}
{ }
Mills et al. 2003
Temperature DependenceSynaptotagmin C2A domain and Calcium
Time (min)
Ca2+ / Synaptotagmin C2A
kca
l/m
ol
Ca2
+c
al/s
10 °C
25 °C
-5
0
5
10
0 30 60 90 120 150 180
0 2 4 6 8
0
1
2
Two calcium binding sites per C2A domain
No robust fit for two site model
10°C 25°C
N 1.8 2.1
KD (M) 450 340
H (cal/mol) +3080 +1830
0
10
0 30 60 90 120 150 180
0 2 4 6 8
0
1
2
Temperature DependenceSynaptotagmin C2A domain and Calcium
Time (min)
Ca2+ / Synaptotagmin C2A
kca
l/m
ol
Ca2
+c
al/s
10 °C
25 °C
37 °C
10°C 25°C 37°C
N1 1.8 2.1 0.9
KD1 (M) 450 340 103
H1 (cal/mol) +3080 +1830 -530
N2 0.9
KD2 (M) 410
H2 (cal/mol) +3770
At higher temperature the reaction is more exothermic
At 37°C the two sites can be fitted and resolved
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
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