binding properties and stoichiometry of the t cell receptor · binding properties and stoichiometry...

Binding Properties and Stoichiometry of the T Cell

Receptor

Jennifer StoneSummer School on Theoretical and

Experimental ImmunologySeptember 2010

Outline

• T Cell Receptor Complex− Components, Assembly, and Organization

• Ligand binding measurements− Surface Plasmon Resonance− Peptide-MHC Multimers− In Situ 2D Binding Measurements

Components of the TCR Complex

α βε γδ ε

CD4

LCK

APC

Class II MHC-peptide

α βε γδ ε

CD4

LCK

APC

Class II MHC-peptide

α βε γδ ε

CD8

APC

Class I MHC-peptide

LCK

α βε γδ ε

CD8

APC

Class I MHC-peptide

α βε γδ ε

CD8

APC

Class I MHC-peptide

LCK

T cell recognition of peptide-MHC ligands

Rudolph et. al. Annu Rev Biophys Biomol Struct 2002

Structures of MHC, TCR, and co-receptors

Cochran et. al. Trends Biochem Sci 2001

The T Cell Receptor Complex

No complete structure of the TCR complex has been solved

Stoichiometry and surface expression/distribution of subunits still a subject of study

Assembly of the TCR-CD3 complex.

Wucherpfennig K W et al. Cold Spring Harb Perspect Biol 2010©2010 by Cold Spring Harbor Laboratory Press

Alarcon et. al. EMBO Rep 2006

Characterization of TCR-CD3 complex from T cells

Early evidence for a Bivalent TCR

Fernandez-Miguel et. al. PNAS 1999

Double TCR-Tg mice

145

83

6050

35 kDa

Early evidence for a Bivalent TCR


Double TCR-Tg mice

Donor quencing of FITC detection

Model of Bivalent TCR


MβCD

Schamel et. al. J Exp Med 2005

Cholesterol-dependent TCR multimers

BN-PAGE : proteins labeled with Coomassie Blue G-250

Doesn’t disrupt membrane protein interactions like SDS

Campi et. al. J Exp Med 2005

TCR microclusters are present before activation

Kuhns et. al. PNAS 2010

TCR complex dimerization

Intracellular ErythropoetinReceptor Signaling Domain Dimerization Assay

Outline



de Mol and Fischer, & Schuck and Zhao, Methods Mol Biol, 2010

A+B AB

Issues to be concerned about:

Temperature

Mass Transport artifacts

Surface heterogeneity

Decay of immobilized analyte

Multivalency or aggregation of solution-phase ligand

Affinity ranges:

very high (pM) affinities can be difficult to measure

estimate only above ~50 µM

t½ ranges

qualitative only under 5-10 seconds

extremely long dissociation can be problematic

Surface Plasmon Resonance

][])[]]([[][max

ABkABABAkdtABd

offon−−=

0 10 20 30 40 50 60 70 80 90-7

-6

-5

-4

-3

t1/2 (s)

KD (M

)

Agonist ▲ Weak Agonist▼ Antagonist

Stone et. al. Immunology 2009

Correlation of binding parameters with stimulatory capacity of interaction

Relatively few peptide-MHC/TCR

interactions measured by SPR

Furthermore, most SPR measurements are taken at 25°C,

while T cell activation occurs at

37°C

T cell activation correlates with KDand t½

Chervin, Stone et. al. J Immunol 2009

Antagonist threshold

1

10

100

0.1

0.01

dEV8

SIYAffi

nity

(KD, µ

M)

Agonist threshold

CD4/CD8 requirement threshold

Optimal activity plateau

dEV8

SIY

Null peptides

Wild-type TCR (2C) High Affinity TCR (2Cm33)

Null peptides

Different Similar

Spectrum of peptide similarity to wild type:

Antigen Specificity Fine Specificity

Antagonist threshold

1

10

100

0.1

0.01

dEV8

SIYAffi

nity

(KD, µ

M)

Agonist threshold

CD4/CD8 requirement threshold

Optimal activity plateau

dEV8

SIY

Null peptides

Wild-type TCR (2C) High Affinity TCR (2Cm33)

Null peptides

Different Similar



Different Similar




Correlation of KD with T cell stimulation

Comparison of wild-type and high-affinity TCR

Kinetic Proofreading model

Short t½ would not be predicted to allow full zeta phosphorylation

Longer t½ would be predicted to be fully phosphorylated and cause T cell activation

Jones et. al., J Immunol, 2008

Kersh et. al., Science, 1998

0 10 20 30 40 50 60 70 80 90-7

-6

-5

-4

-3

t1/2 (s)

KD (M

)

Agonist ▲ Weak Agonist▼ Antagonist


Longer t½ does not always result in better stimulation

Extended t½ results in less potent

stimulation in the case of the OT-1 TCR binding to

OVA(G4)/Kb

Optimal Dwell-time model

Kalergis et. al., Nat Immunol, 2001

Peptide-MHC/TCR interactions with intermediate t½ are the most potent

Based on the hypothesis of serial triggering Valitutti et. al., Nature, 1995

*Cells with engineered TCRs with t½ 100- to 1000-fold longer than wild-type are efficiently and sensitively triggered

Molecular Flexibility (∆Cp)

Rigid body adjustments

Alterations to reach transition state

Alterations to attain fully bound complex

Qi et. al., PNAS, 2006


Krogsgaard et. al., Mol Cell, 2003

A

OOO KRTSTHG ln−=∆−∆=∆

aassockRTEa ln−=

ddisskRTEa ln−=

Measure ∆Cp by SPR or Isothermal Titration Calorimetry

)ln()(O

O

p

O

TO

O

p

O

T

O

T TTCTSTTTCHG

OO∆−∆−−∆+∆=∆

Boniface et. al., PNAS, 1999


]exp[3

2/1

2

2/1CpBAtt DD ∆−=

Qi et. al., PNAS, 2006

Peptide-MHC Confinement Time

Aleksic, Dushek, et. al. Immunity 2010



)k(kkk

T1k

*

on

off

C

*

off

−

−

+==*

*-

-



correlation KD-koff

koff

KD

molecular flexibility

molecular flexibility+confinement time

confinement time

Outline



Casalegno-Garduño et. al. Cancer Immunol Immunother 2009

Peptide-MHC Multimers

IgG or Fcfusion (dimer)

Ultimer(hexamer)

Pentamer

X-Link (dimer-octamer)

Streptavidin-linked Tetramer

Cochran et. al. Trends Biochem Sci 2001


KX KX KXKD

Complications:

Relatively high threshold of sensitivity (up to 10% receptors bound)

“Steady state” measurements do not represent a true equilibrium

Binding affected by multiple factors, including co-receptor

*this may be an advantage

Binding of peptide-MHC multimers to T cells


TCR panel with various binding parameters

TCR t½ (s) KD (nM) S51 A 86 15

m33 46 16 Y26 A 50 17 N27βA 33 40 Y49 A 58 47

S51 A/Y48βA 11 540 Y48βA 2 2900 N30βA 3 8200 Y50 A 0.5 7000

2C 0.9 36,000


Peptide-MHC tetramer t½,tet values


Are KD,tet values valid?

Wooldridge et. al. Immunology 2009

TCR KD: 30 80 >250µM

Co-receptor and peptide-MHC tetramers

The effect of CD8 co-

receptor binding on

MHC tetramer

dissociation

Wooldridge et. al. J Biol Chem 2005

Outline



Toletino et. al. Biophys J 2008

2D Rates of binding and diffusionFRAP: Fluorescence Recovery After Photobleaching

Wu et. al. Biophys J 2008

lipid only

CD2: CD58

CD16aGPI

-RbIgG

2D Rates of binding and diffusion

Wu et. al. Biophys J 2008

2D Rates of binding and diffusion

Rapid initial phase of recovery due to diffusion of unbound molecules

Slower second phase of recovery due to unbinding, diffusion, and re-binding for reaction-limited kinetics

1.4x1041.4x1061.20.1405In vitroIn situIn vitroIn situIn vitroIn situ

KD (µM) t½ (s-1) kon (M-1s-1)

Huppa et. al. Nature 2010

2D Rates of binding and diffusionIn Situ FRET: Fluorescence Resonance Energy Transfer

Huang et. al. Nature 2010

mrmlAcKa and koff Ackon = AcKa×koff

2D Rates of binding and diffusionIn Situ Cell Adhesion Assay – controlled contact time and area

Huang et. al. Nature 2010

Correlation of 2D Binding Parameters with T Cell

Recognition

2D off-rates up to 8300-fold

faster than 3D off-rates

Broader dynamic range of values seen

Reference List• Rudolph MG, Luz JG, Wilson IA. Structural and thermodynamic correlates of T cell signaling. Annu Rev Biophys Biomol

Struct. 2002;31:121-49.

• Cochran JR, Aivazian D, Cameron TO, Stern LJ. Receptor clustering and transmembrane signaling in T cells. Trends BiochemSci. 2001 May;26(5):304-10.

• Wucherpfennig KW, Gagnon E, Call MJ, Huseby ES, Call ME. Structural biology of the T-cell receptor: insights into receptor assembly, ligand recognition, and initiation of signaling. Cold Spring Harb Perspect Biol. 2010 Apr 1;2(4):a005140.

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• Schuck P, Zhao H. The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing. Methods Mol Biol. 2010;627:15-54.

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Reference List• Qi S, Krogsgaard M, Davis MM, Chakraborty AK. Molecular flexibility can influence the stimulatory ability of receptor-ligand

interactions at cell-cell junctions. Proc Natl Acad Sci U S A. 2006 Mar 21;103(12):4416-21.

• Boniface JJ, Reich Z, Lyons DS, Davis MM. Thermodynamics of T cell receptor binding to peptide-MHC: evidence for a general mechanism of molecular scanning. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11446-51.

• Krogsgaard M, Prado N, Adams EJ, He XL, Chow DC, Wilson DB, Garcia KC, Davis MM. Evidence that structural rearrangements and/or flexibility during TCR binding can contribute to T cell activation. Mol Cell. 2003 Dec;12(6):1367-78.

• Aleksic M, Dushek O, Zhang H, Shenderov E, Chen JL, Cerundolo V, Coombs D, van der Merwe PA. Dependence of T cell antigen recognition on T cell receptor-peptide MHC confinement time. Immunity. 2010 Feb 26;32(2):163-74.

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• Wooldridge L, van den Berg HA, Glick M, Gostick E, Laugel B, Hutchinson SL, Milicic A, Brenchley JM, Douek DC, Price DA, Sewell AK. Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface. J Biol Chem. 2005 Jul 29;280(30):27491-501.

• Tolentino TP, Wu J, Zarnitsyna VI, Fang Y, Dustin ML, Zhu C. Measuring diffusion and binding kinetics by contact area FRAP. Biophys J. 2008 Jul;95(2):920-30.

• Wu J, Fang Y, Zarnitsyna VI, Tolentino TP, Dustin ML, Zhu C. A coupled diffusion-kinetics model for analysis of contact-area FRAP experiment. Biophys J. 2008 Jul;95(2):910-9.

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• Huang J, Zarnitsyna VI, Liu B, Edwards LJ, Jiang N, Evavold BD, Zhu C. The kinetics of two-dimensional TCR and pMHCinteractions determine T-cell responsiveness. Nature. 2010 Apr 8;464(7290):932-6.

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