1. Binding Studies on Trafficking Proteins Using Microcalorimetry
McMahon lab
Neurobiology Division
Laboratory of Molecular Biology
Cambridge

2. Clathrin Mediated Endocytosis
Binding
Recruitment
Coating
Receptor
Budding
Uncoating
Clathrin-mediated endocytosis is a highly regulated process which is controlled by multiple protein-protein interactions. The concentration of cargo molecules, the recruitment of clathrin and the invagination of the membrane are mediated by the AP adaptor complex and other adaptor proteins.
Ligand
AP adaptor complex
Regulatory adaptor
Clathrin
Dynamin

3. Receptor Mediated Endocytosisb c
Clathrin-coated pits and vesicles can be visualised by electron microscopy.
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)

4. AP Adaptor Complex Appendage 1-4 , , ,  Hinge Trunk 1-4 1-4
Collins et al. 2002
Appendage
binds regulators
Hinge
binds clathrin
Trunk
binds lipids and
membrane proteins
1-4
1-4
, , , 
1-4
The adaptor protein (AP) complexes are heterotetrameric proteins. There are four different AP complexes in higher eucaryots. The AP complex can be divided in three parts: the trunk, the hinge and the appendage domains. While the trunk is involved in cargo and lipid binding, the hinge region binds clathrin and the C-terminal appendage domains of the two large subunits interact with accessory proteins such as other adaptor proteins and lipid modifying enzymes etc.
AP-1 (): TGN / Endosome
AP-2 (): Plasma membrane
AP-3 (): Lysosome
AP-4 (): TGN

5. AP Trafficking Pathways
Plasma membrane
AP-2
Endosome
Lysosome
AP-3
Lysosome-related
Organelle
Each of the four AP complexes is involved in one or more specific trafficking pathways.
AP-4
GGA
AP-1
AP-3
Trans-Golgi-Network

6. AP Appendage Domains -Adaptin -Adaptin -Adaptin DP(F/W) FxDxF
DPW
FxxF
The appendage domains contain several binding sites for short peptide motifs which occur in the accessory proteins.
-Adaptin
-Adaptin
-Adaptin
Owen et al. 2000
Owen et al. 1999
Brett et al. 2002
Kent et al. 2002
Nogi et al. 2002

7. Regulatory Adaptors DOMAIN PARTNER Epsin1 EpsinR AP180 Dab2 Eps15
Amphiphysin1
Aminoacid
250
1000
500
750
DOMAIN PARTNER
SH3 PxxPxR
PTB Receptor and Lipids
ANTH/ENTH Lipids
BAR Lipids
EH NPF
Clathin-Box Clathrin
DxF/W - and -Adaptin
NPF EH
PxxPxR SH3
These peptide motifs are usually found in high copy numbers in the Motif Domains (MDs) of accessory proteins leading to strong binding to assembled AP complexes.

8. Interactions in Trafficking
Amphiphysin
Receptor
Lipids
FEDNF
Yxx
or LL
AP-Complex
LLDLD
Because of all the possible interactions between endocytic proteins they can form a complex interaction network.
DxF or
FxxF
LLDLD
DxF or FxxF
NPF
Epsin1
EpsinR
AP180
Dab2
Eps15
Clathrin
LLDLD

9. Determination of Binding Constants
Definition of Association and Dissociation Constants:
k1 [P]free = conc. of free protein
For 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)
The dissociation constant is a measure of the strength of an interaction. It can be defined either by the equilibrium between the binding and dissociation reactions or between bound and free ligand.

10. 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
A simple binding model can be described by a hyperbolic curve. In this model the binding sites of the protein have an occupancy of 50 % if the ligand concentration is equal to the dissociation constant.
Please note that this model is only correct if the concentration of free ligand is very similar to the concentration of total ligand, i.e. if the protein concentration is much lower than the ligand concentration. If this is not the case, a quadratic binding model has to be applied.

11. Methods to determine Binding Constants
Signal Information Advantage Disadvantage
Spectroscopy change of absorption KD ( M) in solution probe needed
(Fluorescence, UV/Vis, CD) or emission of light
Microcalorimetry heat of binding KD ( M) no labels, large sample
H, S, n in solution
direct access to H
direct access to n
Surface Plasmon Resonance change of refractive KD ( M) small sample, surface coupled,
index due to mass k1, k-1 automated ligand must have
large mass
Stopped-Flow coupled to spectroscopy KD ( M) fast probe needed
k1, k-1
Analytical Ultracentrifugation absorption at different KD ( M) good for slow
radii for different times homomeric
interactions
Nuclear Magnetic Resonance shift of magnetic KD ( M) in solution, slow,
resonance frequency structural large sample,
information expensive
Binding Assays various, e.g. SDS-PAGE, KD ( M) can be most sometimes
densitometry, radio- sensitive inaccurate
activity
There are several methods to measure the interaction between biological molecules. Each one has certain advantages and limitations.

12. Isothermal Titration Calorimetry (ITC)
In isothermal titration calorimetry (ITC), the protein in the measuring cell is kept at a constant temperature. The ligand is added stepwise from a syringe and the heat which is released or taken up due to binding is compensated by a sensitive thermostat. The electrical power of the thermostat is directly related to the enthalpy of the reaction.

13. Isothermal Titration Calorimetry (ITC)
Picture of a modern ITC device
Taken from Micro Cal website

14. 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= 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?
The high sensitivity of the ITC devices allows the precise measurement of binding reactions over a broad range of concentrations.

15. 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= 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?
 kcal / mol

16. 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= 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?
 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)

17. Isothermal Titration Calorimetry (ITC)
Ligand / Protein
cal/s
kcal/mol Ligand
Time (min)
stochiometry: N
enthalpy: H
affinity: 1/Kd
A standard ITC trace consists of two panels. The upper panel shows the heat trace of the thermostat over the time of the experiment with the individual injections of ligand as peaks. By integrating the area of the peaks and plotting them against the molar ratio of ligand and protein one obtains the points depicted in the lower panel. By fitting a quadratic binding curve to the data, one obtains the binding isotherm. From the shape of this curve one can easily estimate the enthalpy and stochiometry of the reaction as well as the affinity (which is the inverse of the dissociation constant). These are the parameters of the fit algorithm and are also used to calculate the entropy of the reaction.

18. Isothermal Titration Calorimetry (ITC)
Ligand / Protein
cal/s
kcal/mol Ligand
Time (min)
stochiometry: N
enthalpy: H
affinity: 1/Kd

19. a-Adaptin and Amphiphysin
Binding Specificity
a-Adaptin and Amphiphysin
DNF+DPF-SGA
Amph
DNF-SGA
DPF-SGA
Extract
-Adaptin
-Adaptin
Amph
DNF-ANF
DNF-DPF
DNF-RPF
DNF-DPP
DNF-DPW
DNF-DGF
DNF-DLF
DNF-DAF
DNF-DDF
DNF-DSF
DNF-EPL
DNF-DIF
Extract
-Adaptin
-Adaptin
The protein amphiphysin contains two DxF/W motif which bind to the alpha appendage domain of the AP2 complex. Western blots have shown, that the FEDNF motif is the major binding site.
Sequence
rAmphiphysin1 INFFEDNFVPEINVTTPSQNEVLEVKKEE TLLDLDFDPFKPDVTPAGSAAATHSPMSQTLPWDLW
rAmphiphysin2 LSLFDDAFVPEISVTTPSQFEAPGPFSEQASLLDLDFEPLPPVASPVKAPTPSG QSIPWDLW
Praefcke et al. 2004
Olesen et al. 2007

20. a-Adaptin and Amphiphysin
Binding Specificity
a-Adaptin and Amphiphysin
DNF-Peptide / -Appendage
Time (min)
cal/s
kcal/mol Peptide 12 7 8
DxF Peptide Sequence KD (M)
DNF 7mer FEDNFVP
DNF to RNF 7mer FERNFVP no binding
DNF 8mer FEDNFVPE
DNF 12mer INFFEDNFVPEI
DNF to DPF 12mer INFFEDPFVPEI
DNF to DAF 12mer INFFEDAFVPEI
DNF FE-change INFEFDNFVPEI
DPF 12mer LDLDFDPFKPDV
DPF to DNF-12mer LDLDFDNFKPDV no binding
This was confirmed by ITC where peptides corresponding to the individual binding sites were used as ligands. The FEDNF binds almost 100 fold stronger than the DPF motif.
Sequence
rAmphiphysin1 INFFEDNFVPEINVTTPSQNEVLEVKKEE TLLDLDFDPFKPDVTPAGSAAATHSPMSQTLPWDLW
rAmphiphysin2 LSLFDDAFVPEISVTTPSQFEAPGPFSEQASLLDLDFEPLPPVASPVKAPTPSG QSIPWDLW
Praefcke et al. 2004
Olesen et al. 2007

21. a-Adaptin and Amphiphysin
Binding Specificity
a-Adaptin and Amphiphysin
DNF-Peptide / -Appendage
Time (min)
cal/s
kcal/mol Peptide 12 7 8
DxF Peptide Sequence KD (M)
DNF 7mer FEDNFVP
DNF to RNF 7mer FERNFVP no binding
DNF 8mer FEDNFVPE
DNF 12mer INFFEDNFVPEI
DNF to DPF 12mer INFFEDPFVPEI
DNF to DAF 12mer INFFEDAFVPEI
DNF FE-change INFEFDNFVPEI
DPF 12mer LDLDFDPFKPDV
DPF to DNF-12mer LDLDFDNFKPDV no binding
Synaptojanin LDGFEDNFDLQS
HIP1 DNKFDDIFGSSF
Dab2 QSNFLDLFKGNA 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

22. Lipid Binding Epsin1 ENTH domain Ford et al. 2002 No Liposomes
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 Liposomes
PtdIns(3,4,5)P3
P S P S P S P S P S P S P S
PtdIns(3)P
PtdIns(4)P
PtdIns(3,4)P2
PtdIns(4,5)P2
PtdIns(3,5)P2
The epsin proteins contain an N-terminal ENTH domain which interacts with phosphoinositols. The specificity for certain phospholipid could be measured by liposome cosedimentation assays and by lipid overlay blots.
Ford et al. 2002

23. Lipid Binding Lipid Binding Epsin1 ENTH domain Time (min)
KD (M)
 Ins(1,4)P2 >1,000
 Ins(1,5)P >1,000
 Ins(1,3,5)P3 120
 Ins(1,4,5)P
 Ins(1,3,4,5)P
 InsP
diC8PtdIns(4,5)P
cal/s
kcal/mol InsPx
A quantitative measurement with inositolphosphates as well as with short-chain lipids revealed a high preference for PI(4,5)P2 and PI(3,4,5)P3 in the case of epsin1.
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

24. Lipid Binding Lipid Binding Epsin1 ENTH domain Time (min) Liposomes
cal/s
kcal/mol Protein
Epsin1
Disabled2
Protein / PI(4,5)P2 in outer leaflet
ENTH domains do not only bind lipids but also induce curvature into membranes. This can be seen in electron microscopy of tubulated liposomes. In ITC this process can be directly followed and compared to lipid binding proteins which do not induce curvature such as the PTB domain of disabled-2.
Data for Epsin1-ENTH with liposomes is different from control protein
 ITC reveals tubulation of liposomes by the ENTH domain
Ford et al. 2002

25. Multiple Binding Sites
D325R
D328R
D349R
D371R
E391R
D422R
Multiple Binding Sites
EpsinR and -Adaptin
Truncations Point Mutations
Clathrin
-Adaptin
The epsin1 related protein epsinR binds specifically to the gamma-appendage domain of the AP1 complex. Western blots revealed the presence of two strong binding sites around the two DxF motifs.
<325
<328
<334
<345
<349
(291)AHYTGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS
FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426)
<371
<379
<391
<397
<422
Mills et al. 2003

26. Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
cal/s
kcal/mol EpsinR
EpsinR / -Adaptin-Appendage
One Site Model
N KD (M)
Two Site Model
N1 KD (M)
N2 KD (M)
Mills et al. 2003

27. Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
Time (min)
cal/s
cal/s
kcal/mol EpsinR
kcal/mol -Adaptin
EpsinR / -Adaptin-Appendage
The presence of two binding sites was confirmed by ITC. Depending on the setup of the experiment, i.e. swapping the proteins in the cell and syringe, the reaction showed either a stochiometry of 0.5 (left side) or two consecutive reactions with stochiometries around 1.0 (right side). The fitting software can be adjusted accordingly and yield comparable binding constants. However, fitting of six parameters can be difficult especially if the two dissociation constants are similar.
-Adaptin-Appendage / EpsinR
One Site Model
N KD (M)
Two Site Model
N1 KD (M)
N2 KD (M)
One Site Model
N KD (M)
1.3 19
Two Site Model
N1 KD (M)
N2 KD (M)
swap cell and syringe content
Mills et al. 2003

28. Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
Peptide KD (M)
EpsinR -Adaptin
P1-SGDLVDLFDGTS no binding
P2-TGGSADLFGGFA
P3-SADLFGGFADFG
P4-FGGFADFGSAAA > 220
P5-TSGNGDFGDWSA P3
cal/s P5
kcal/mol Peptide P3 P5
EpsinR Peptide / Adaptin-Appendage
Experiments with overlapping peptides showed slightly lower affinities. Nevertheless, the two binding motifs could be mapped. P1 P3 P4 P2 P5
291(AHY)TGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS
FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426)
Mills et al. 2003

29. Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
Peptide KD (M)
EpsinR -Adaptin
P1-SGDLVDLFDGTS no binding
P2-TGGSADLFGGFA
P3-SADLFGGFADFG
P4-FGGFADFGSAAA > 220
P5-TSGNGDFGDWSA
-Synergin
PEEDDFQDFQDA
Eps15
SFGDGFADFSTL
Epsin1
EPDEFSDFDRLR
EF-hand
NEDDFGDFGDFG P3 P5
cal/s
Sy
kcal/mol Peptide P3 P5
Sy
EpsinR Peptide / Adaptin-Appendage
A comparison with other gamma-adaptin binding proteins led to the identification of a novel, AP1 specific binding motif (FxxF/W).
<349
291(AHY)TGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS
FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426) P3
<371 P5
Mills et al. 2003

30. Multiple Binding Sites
EpsinR and -Adaptin
Time (min)
Peptide KD (M)
EpsinR -Adaptin
P1-SGDLVDLFDGTS no binding
P2-TGGSADLFGGFA
P3-SADLFGGFADFG
P4-FGGFADFGSAAA > 220
P5-TSGNGDFGDWSA
-Synergin
PEEDDFQDFQDA
Eps15
SFGDGFADFSTL
Epsin1
EPDEFSDFDRLR
EF-hand
NEDDFGDFGDFG P3 P5
cal/s
Sy
kcal/mol Peptide P3 P5
Sy
EpsinR Peptide / Adaptin-Appendage
A comparison with other gamma-adaptin binding proteins led to the identification of a novel, AP1 specific binding motif (FxxF/W).
EpsinR contains two binding sites for -Adaptin
Identification of consensus motif using peptides
Motif is also present in other trafficking proteins

31. Exothermic Decrease in Entropy { } Mills et al. 2003
{ }
Exothermic
Decrease
in Entropy
Except in{..}
Using site-directed mutagenesis, each binding site could be eliminated.
Mills et al. 2003

32. Temperature Dependence Synaptotagmin C2A domain and Calcium
Time (min)
10°C 25°C N
KD (M)
H (cal/mol)
10 °C
cal/s
25 °C
kcal/mol Ca2+
The protein synaptotagmin is involved in the exocytosis of synaptic vesicles. It is able to interact with lipid membranes in presence of calcium ions. The C2A domain of synaptotagmin1 binds two calcium ions. The endothermic binding reaction can be measured in the ITC.
Ca2+ / Synaptotagmin C2A
Two calcium binding sites per C2A domain
No robust fit for two site model

33. Temperature Dependence Synaptotagmin C2A domain and Calcium
Time (min)
10°C 25°C 37°C N
KD1 (M)
H1 (cal/mol) N
KD2 (M) 410
H2 (cal/mol)
10 °C
cal/s
25 °C
37 °C
kcal/mol Ca2+
At physiological temperature, the two binding reactions display different thermodynamic behaviour. While the high affinity binding site is still endothermic, the low affinity site is now exothermic, leading to a wave-like shape of the binding isotherm.
Ca2+ / Synaptotagmin C2A
At higher temperature the reaction is more exothermic
At 37°C the two sites can be fitted and resolved

34. 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