recognition

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Lecture9

Molecularrecognition

AntoinevanOijen

BCMP201Spring2008

Structuralprinciplesofbinding

Lastweek:Kineticsandthermodynamicsofbinding

Today:Structuralprinciplesofbinding

4fundamentalfunctionsofproteins:

1)Binding

2)Catalysis

3)Switching

4)Structural

Allinvolvebinding!

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Thenatureofprotein-proteininteractions

Bindingenergysimplysumofenergiesof

-hydrogenbonds

-ioniccontacts

-vanderWaalscontacts?

Thenatureofprotein-proteininteractions

ChothiaandJanin(1975,Nature):

Electrostatic,H-bond,vdWinteractionsarealsoprentinsolution;

formingthematthebindinginterfacedoesn’treduceΔG0

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Thenatureofprotein-proteininteractions

Maindeterminantofbindingenergyishydrophobicity(entropiceffect)

Thenatureofprotein-proteininteractions

Bindingenergyincreaswithareaofinterface

Maximum~0.1kJ/molperÅ2interactionsurface

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Specificity

Highspecificityinterfacesmustbehighlycomplementary

Specificityprovidedby:

1)Ioniccomplementarity

2)Hydrogenbondcomplementarity

3)Stericcomplementarity(vanderWaals)

Example:ahormone-receptorinterface

humanGrowthHormone-humanGrowthHormonereceptor

90°

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Example:ahormone-receptorinterface

K

D

ofhGH-hGHrinteraction=0.3nM

Using:

!

K

D

=e("G0/RT)

ΔG0=-54kJ/mol(=12.7kcal/mol)

(R=8.3J-1mol-1K-1

T=295K)

~1300Å2buriedsurface

Alaninescanningmutagenesis

SystematicallymutateeachofthesurfaceresiduesintoAlanine

(Clacksonetal.,Science(1995);267,383)

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Example:ahormone-receptorinterface

‘Hotspots’:

W104AandW169AeachincreaΔG0by

>4.5kcal/mol(~19kJ/mol)

EachincreasK

D

from0.3nMto>0.7µM

(Clacksonetal.,Science(1995);267,383)

Example:ahormone-receptorinterface

Functional

epitope

Structural

epitope

Compareinteractionsurfacewith

cross-ctionthroughglobularprotein:

hydrophobiccore,hydrophilicexterior

(Clacksonetal.,Science(1995);267,383)

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Example:ahormone-receptorinterface

ComplementarityoffunctionalepitopesofGHandGHr

(Clacksonetal.,Science(1995);267,383)

Lockinkeyversusinducedfit

‘Lockinkey’

‘Inducedfit’

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Inducedfit

1)Surfacesidechainscanmove

2)Surfaceloopscanmove

3)Domainscanmoveathinges

Helix-turn-helixmotifs

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Lacrepressor

Lacrepressor

FoldinguponbindingtoDNA

(Kalodimotal.,Science(2004),305,386)

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Lacrepressor

Foldingonlyuponbindingto

specificDNA

Nonspecificcomplexformedby

electrostaticinteractions

(Kalodimotal.,Science(2004),305,386)

Amechanismfortargetlocation?

(sicalJrnl.(2004);87,4021)

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Antibodies

Immunoglobulins/antibodies:

SecretedbyBcellstobindtoantigens

Constantdomains(C)

Variabledomains(V)

(Figuresfrom:Branden,Tooze;IntroductiontoProteinStructure)

Immunoglobulins

Regionsinvariabledomainsshowhypervariability

(complementaritydeterminingregions;CDR)

(Figurefrom:Branden,Tooze;IntroductiontoProteinStructure)

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Antigen-bindingsite

Antigen-bindingsiteisformedbycloassociationofthe

hypervariableregionsfrombothheavyandlightchains

Lightchain

Heavychain

Antigen-bindingsite

Lightchain

Heavychain

Antigen-bindingsiteisformedbycloassociationofthe

hypervariableregionsfrombothheavyandlightchains

(Figuresfrom:Branden,Tooze;IntroductiontoProteinStructure)

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Inducedfit

InducedfituponbindingofanHIV-1peptidetoFabfragmentofIgG

Wilsonetal.,Structure(1993);1,83-93

Domainflexibility

Domainflexibilitycangiveritodramatic

increainbindingaffinity:divalentbinding

virus

IgG

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Cooperativeassociation

virus

IgG

K

D

Fab≈10-6M;WhatisK

D

IgG?

1)CalculateΔG0

Fab

=RTlnK

D

=-34kJ/mol

2)Multiplyby2for2bonds:-68kJ/mol

3)Plusanentropicfactorof~-25kJ/mol

4)ΔG0

IgG

=-93kJ/molK

D

IgG=3x10-17M!!

Entropypenaltyisonlypaidonce!

ImmunoglobulinfoldinMHCandT-cellreceptors

Antibody-antigenHighaffinity,highspecificity

MHC-peptideHighaffinity,lowspecificity

T-cellreceptor-MHC/peptideLowaffinity,highspecificity

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Antibodytechniques

(Lehninger,PrinciplesofBiochemistry)

Take-homemessages

1)Protein-proteininteractionsaremainlymediatedbyhydrophobiceffects

2)Surfacecomplementaritycontributestospecificity

3)Lock-in-keyversusinducedfit