Binding Studies on Trafficking Proteins Using Microcalorimetry McMahon lab

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

2 Clathrin Mediated Endocytosis Binding Recruitment Coating Budding Uncoating Receptor Ligand AP adaptor complex Regulatory adaptor Clathrin Dynamin

3 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)

4 AP Adaptor Complex β 1-4 α, γ, δ, ε Appendage binds regulators Hinge binds clathrin µ 1-4 σ 1-4 Trunk binds lipids and membrane proteins AP-1 (γ): TGN / Endosome AP-2 (α): Plasma membrane AP-3 (δ): Lysosome AP-4 (ε): TGN Collins et al. 22

5 AP Trafficking Pathways Plasma membrane AP-2 Endosome Lysosome AP-3 Lysosome-related Organelle AP-4 GGA AP-1 AP-3 Trans-Golgi-Network

6 AP Appendage Domains FxDxF DP(F/W) DP(F/W) DPW FxxF α-adaptin β-adaptin γ-adaptin Owen et al Brett et al. 22 Owen et al. 2 Kent et al. 22 Nogi et al. 22

7 Regulatory Adaptors Epsin1 EpsinR AP18 Dab2 Eps15 Amphiphysin1 Aminoacid DOMAIN SH3 PTB ANTH/ENTH BAR EH Clathin-Box DxF/W NPF PxxPxR PARTNER PxxPxR Receptor and Lipids Lipids Lipids NPF Clathrin α- and β-adaptin EH SH3

8 Interactions in Trafficking Amphiphysin Receptor Lipids YxxΦ or LL LLDLD AP-Complex DxF or FxxF Clathrin Eps15 LLDLD NPF Epsin1 EpsinR AP18 Dab2

9 Determination of Binding Constants Definition of Association and Dissociation Constants: k 1 [P] f ree = conc. of free protein For a binding reaction at equilibrium: P + L PL [L] f ree = conc. of free ligand k -1 [PL] = conc. of PA complex k 1 = rate constant for formation of [PL] k -1 = rate constant for breakdown of [PL] The rate of formation of [PL] is k 1 [P] f ree [L] f ree, where k 1 is a second order rate constant with units of l/mol -1 s -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 k 1 [P] f ree [L] f ree = k -1 [PL]. Also recall that: K D = k -1 / k 1 = [P] f ree [L] f ree / [PL] = 1 / K A K D 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] f ree + [PL]) = [L] f ree / ([L] f ree + K D ) Use [PL] = K A [P] f ree [L] f ree Divide through by K A Replace K A by 1 / K D

10 Determination of Binding Constants Special cases: y = [L] f ree / ([L] f ree + K D ) For [L] f ree = : y = For [L] f ree : y = 1 For [L] f ree = K D : y =.5 nothing bound full occupancy 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

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

12 Isothermal Titration Calorimetry (ITC)

13 Isothermal Titration Calorimetry (ITC) Taken from Micro Cal website

14 Isothermal Titration Calorimetry (ITC) Review of Free Energies, Enthalpies, and Entropies of Binding G bind = RT lnk D (where R= 1.98 cal mol 1 K -1 ; T= K, and RT =.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 1-fold change in binding constant corresponds to 1.4 kcal / mol. G A1-A2 = RT ln(k D A1 / K D A2)= (.62 kcal / mol)ln(1-8 M / 1-7 M) = -1.4 kcal / mol How many kcal / mol change in free energy do you need to change K D 1-fold?

15 Isothermal Titration Calorimetry (ITC) Review of Free Energies, Enthalpies, and Entropies of Binding G bind = RT lnk D (where R= 1.98 cal mol 1 K -1 ; T= K, and RT =.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 1-fold change in binding constant corresponds to 1.4 kcal / mol. G A1-A2 = RT ln(k D A1 / K D A2)= (.62 kcal / mol)ln(1-8 M / 1-7 M) = -1.4 kcal / mol How many kcal / mol change in free energy do you need to change K D 1-fold? kcal / mol

16 Isothermal Titration Calorimetry (ITC) Review of Free Energies, Enthalpies, and Entropies of Binding G bind = RT lnk D (where R= 1.98 cal mol 1 K -1 ; T= K, and RT =.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 1-fold change in binding constant corresponds to 1.4 kcal / mol. G A1-A2 = RT ln(k D A1 / K D A2)= (.62 kcal / mol)ln(1-8 M / 1-7 M) = -1.4 kcal / mol How many kcal / mol change in free energy do you need to change K D 1-fold? kcal / mol Recall also that free energy has enthalpy and entropy components: G = H -T S (and therefore) RTlnK A = 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) Time (min) µcal/s -2 kcal/mol Ligand affinity: 1/K d stochiometry: N enthalpy: H Ligand / Protein

18 Isothermal Titration Calorimetry (ITC) Time (min) µcal/s -2 kcal/mol Ligand affinity: 1/K d stochiometry: N enthalpy: H G= Ligand / Protein H= 19.9 T S= 5.8

19 Binding Specificity α-adaptin and Amphiphysin Amph DNF-SGA DPF-SGA DNF+DPF-SGA Extract Amph 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 α -Adaptin α -Adaptin β-adaptin β-adaptin Sequence ramphiphysin1 INFFEDNFVPEINVTTPSQNEVLEVKKEE TLLDLDFDPFKPDVTPAGSAAATHSPMSQTLPWDLW ramphiphysin2 LSLFDDAFVPEISVTTPSQFEAPGPFSEQASLLDLDFEPLPPVASPVKAPTPSG QSIPWDLW Praefcke et al. 24 Olesen et al. 27

20 5 7 Time (min) Binding Specificity α-adaptin and Amphiphysin DxF Peptide Sequence K D (µm) µcal/s kcal/mol Peptide DNF-Peptide / α-appendage DNF 7mer FEDNFVP 21 DNF to RNF 7mer FERNFVP no binding DNF 8mer FEDNFVPE 28 DNF 12mer INFFEDNFVPEI 2.5 DNF to DPF 12mer INFFEDPFVPEI 12 DNF to DAF 12mer INFFEDAFVPEI 21 DNF FE-change INFEFDNFVPEI 18 DPF 12mer LDLDFDPFKPDV 19 DPF to DNF-12mer LDLDFDNFKPDV no binding Sequence ramphiphysin1 INFFEDNFVPEINVTTPSQNEVLEVKKEE TLLDLDFDPFKPDVTPAGSAAATHSPMSQTLPWDLW ramphiphysin2 LSLFDDAFVPEISVTTPSQFEAPGPFSEQASLLDLDFEPLPPVASPVKAPTPSG QSIPWDLW Praefcke et al. 24 Olesen et al. 27

21 5 Time (min) Binding Specificity α-adaptin and Amphiphysin 7 DxF Peptide Sequence K D (µm) µcal/s kcal/mol Peptide DNF-Peptide / α-appendage DNF 7mer FEDNFVP 21 DNF to RNF 7mer FERNFVP no binding DNF 8mer FEDNFVPE 28 DNF 12mer INFFEDNFVPEI 2.5 DNF to DPF 12mer INFFEDPFVPEI 12 DNF to DAF 12mer INFFEDAFVPEI 21 DNF FE-change INFEFDNFVPEI 18 DPF 12mer LDLDFDPFKPDV 19 DPF to DNF-12mer LDLDFDNFKPDV no binding Synaptojanin LDGFEDNFDLQS 4.5 HIP1 DNKFDDIFGSSF 1 Dab2 QSNFLDLFKGNA no binding DNF-site is 8 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. 24 Olesen et al. 27

22 Lipid Binding Epsin1 ENTH domain PtdCho PtdEth PtdIns(5)P PtdIns(4)P PtdIns(3)P PtdIns LysoPtdCho LysoPtdAcid Blank PtdSer PtdAcid PtdIns(3,4,5)P 3 PtdIns(3,5)P 2 PtdIns(4,5)P 2 PtdIns(3,4)P 2 Sphing-1-P P S P S P S P S P S P S P S Ford et al. 22

23 Time (min) Lipid Binding Epsin1 ENTH domain kcal/mol InsP x µcal/s K D (µm) Ins(1,4)P 2 >1, Ins(1,5)P 2 >1, Ins(1,3,5)P 3 12 Ins(1,4,5)P Ins(1,3,4,5)P InsP 6.55 dic 8 PtdIns(4,5)P InsP x / Epsin1 ENTH Good correlation between ITC and other binding assays Head groups are a good model for the lipid molecules Ford et al. 22

24 kcal/mol Protein µcal/s Epsin1 Disabled2 Time (min) Protein / PI(4,5)P 2 in outer leaflet Lipid Binding Epsin1 ENTH domain 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. 22

25 Truncations Point Mutations Multiple Binding Sites EpsinR and γ-adaptin D325R D328R D349R D371R E391R D422R Clathrin γ-adaptin (291)AHYTGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS <325 <328 <334 <345 <349 <371 <379 <391 <397 <422 FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426) Mills et al. 23

26 Time (min) Multiple Binding Sites EpsinR and γ-adaptin kcal/mol EpsinR µcal/s EpsinR / γ-adaptin-appendage One Site Model N K D (µm) Two Site Model N1 K D (µm) N2 K D (µm) Mills et al. 23

27 Multiple Binding Sites EpsinR and γ-adaptin Time (min) Time (min) kcal/mol EpsinR µcal/s EpsinR / γ-adaptin-appendage µcal/s kcal/mol γ-adaptin γ-adaptin-appendage / EpsinR One Site Model N K D (µm) Two Site Model N1 K D (µm) N2 K D (µm) swap cell and syringe content One Site Model N K D (µm) Two Site Model N1 K D (µm).9.72 N2 K D (µm) Mills et al. 23

28 µcal/s kcal/mol Peptide P3 P5 P3 P5 Time (min) EpsinR Peptide / Adaptin-Appendage Multiple Binding Sites EpsinR and γ-adaptin Peptide K D (µm) EpsinR γ-adaptin P1-SGDLVDLFDGTS no binding P2-TGGSADLFGGFA 23 P3-SADLFGGFADFG 11 P4-FGGFADFGSAAA > 22 P5-TSGNGDFGDWSA (AHY)TGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS P1 P2 P3 P4 FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426) P5 Mills et al. 23

29 µcal/s kcal/mol Peptide P3 P5 γsy P3 P5 γsy Time (min) EpsinR Peptide / Adaptin-Appendage Multiple Binding Sites EpsinR and γ-adaptin Peptide K D (µm) EpsinR γ-adaptin P1-SGDLVDLFDGTS no binding P2-TGGSADLFGGFA 23 P3-SADLFGGFADFG 11 P4-FGGFADFGSAAA > 22 P5-TSGNGDFGDWSA 48 γ-synergin PEEDDFQDFQDA 13 Eps15 SFGDGFADFSTL 18 Epsin1 EPDEFSDFDRLR 2 EF-hand NEDDFGDFGDFG 8 291(AHY)TGDKASPDQNASTHTPQSSVKTSVPSSKSSGDLVDLFDGTSQSTGGSADLFGGFADFGSAAASGS P3 <371 FPSQVTATSGNGDFGDWSAFNQAPSGPVASSGEFFGSASQPAVELVSGSQSALGPPPAASNSSDLFDL(426) P5 Mills et al. 23 <349

30 µcal/s kcal/mol Peptide P3 P5 γsy P3 P5 γsy Time (min) EpsinR Peptide / Adaptin-Appendage Multiple Binding Sites EpsinR and γ-adaptin Peptide K D (µm) EpsinR γ-adaptin P1-SGDLVDLFDGTS no binding P2-TGGSADLFGGFA 23 P3-SADLFGGFADFG 11 P4-FGGFADFGSAAA > 22 P5-TSGNGDFGDWSA 48 γ-synergin PEEDDFQDFQDA 13 Eps15 SFGDGFADFSTL 18 Epsin1 EPDEFSDFDRLR 2 EF-hand NEDDFGDFGDFG 8 EpsinR contains two binding sites for γ-adaptin Identification of consensus motif using peptides Motif is also present in other trafficking proteins

31 Isothermal Calorimetry Titration with EpsinR N3 cons truct N1 N2 K D 1 K D 2 ² H1 ² H2 T² S1 T² S2 N3 + γ-appendage Average N3 D342R + γ-appendage Average N3 D349R + γ-appendage Average N3 D371R + γ-appendage Average N3 + α-appendage Average N3 D342R + α-appendage β-appendage + N { +173 } GST-GGA1 + N K D (µm) H (cal mol -1 ) T S (cal mol -1 ) Exothermic Decrease in Entropy Except in{..} Mills et al. 23

32 Temperature Dependence Synaptotagmin C2A domain and Calcium Time (min) kcal/mol Ca 2+ µcal/s C 25 C Ca2+ / Synaptotagmin C2A 1 C 25 C N K D (µm) H (cal/mol) Two calcium binding sites per C2A domain No robust fit for two site model

33 Temperature Dependence Synaptotagmin C2A domain and Calcium Time (min) kcal/mol Ca 2+ µcal/s C 25 C 37 C Ca2+ / Synaptotagmin C2A 1 C 25 C 37 C N K D 1 (µm) H1 (cal/mol) N2.9 K D 2 (µm) 41 H2 (cal/mol) +377 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