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

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)


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?







- 2.8 kcal / mol







- 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)



-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


-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



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


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


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


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


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


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


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


Mills et al. 2003


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


Time (min)


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



-SynerginPEEDDFQDFQDA 13Eps15SFGDGFADFSTL 180Epsin1EPDEFSDFDRLR 200EF-handNEDDFGDFGDFG 8

P3

P5

P5

P5


cal

/skc

al/

mo

l P

ep

tid

e

P3

P3

<3

71

<3

49

291(AHY)TGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS


Time (min)

Sy

Sy


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



-SynerginPEEDDFQDFQDA 13Eps15SFGDGFADFSTL 180Epsin1EPDEFSDFDRLR 200EF-handNEDDFGDFGDFG 8

P5

P5


cal

/skc

al/

mo

l P

ep

tid

e

P3

P3

Time (min)

Sy

Sy


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

Documents

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