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“Most complex biological problems can be solved by simply looking at them” - Richard Feynman

Immune recognition comprised of a vast array of diverse receptor/ligand interactions

Fc-R

IgG

IgG

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-p

-protein (HSV gIgE)-non-protein (prenyl-P)-non-classical MHC (t10/t22)-classical MHC (IEk)

Most immune receptors composed of Immunoglobulin fold:

Many variations of Ig-fold topology:

-variations primarily dictated by the number ofresidues between cysteines, and the length of thegap between C and E strands.

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IGG2a

VVLL VVHH

CCHH11

CCHH33

CCHH22

CCLL

VVHH

VVLL CCLL

CCHH11

CCHH33

CCHH22

FVFV

FABFAB

FCFC

VH VL CL CH3 CH1 CH2

VH 100 36 14 13 12 12

VL 100 20 20 17 15

CL 100 42 39 34

CH3 100 38 33

CH1 100 29

CH2 100

NtermNterm

Cterm

SequenceSequenceSimilaritySimilarity

RMSRMSDeviationDeviation

VH VL CL CH3 CH1 CH2

VH 0.00 1.04 1.29 1.63 1.53 1.26

VL 0.00 1.31 1.61 1.32 1.07

CL 0.00 1.12 1.15 0.69

CH3 0.00 1.03 1.23

CH1 0.00 1.01

CH2 0.00

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antibody antibody

antibodyviralantigen

viralantigen

MHCMHC

X Y

Humoral Immunity

XX

Yviralantigen

viralantigen

MHCTCR

CTL

Cell Mediated Immunity

MHC

Kill

NoKill

antibodyFab

Fab Fc

T-cellreceptor

T-cell

- antibody can recognize antigen independent of MHC

- TCR must recognize antigen in the context of MHC

SS

SS

SS

SS

-S-S-

VV

CCα

α β

β

N N

immunoglobulin fold

Assembly:

Variable (V) Joining (J) Constant (C)

Complementarity-Determining Regions (CDR)

Diversity (D)V J C

1 2 3

1 2 3

α

β

Immunoglobulin fold

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-both antibody and TCR assemble by stepwise recombination of V-D-J and C-regions.-the TCR is analogous to an Fab of an immunoglobulin.-TCR and Fab composed of four Ig-domains whose loopsare positioned to form the antigen binding site.-the spatial disposition of each of these loops are similar butnot identical. These differences have relevance to thedifferent modes of antigen recognition.

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•Antibody has at least three modes of inter-domain variability. The VH/VL rotational variation impacts on antigenrecognition.•The great variability (~150 degrees) in the “elbow angle” has no bearing an antigen recognition.

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The TCR has no variability in elbow angle, likely linked to TCR as a signaling receptor.

α β receptorsImmunoglobulinElements

-1016-1016Total diversity

-1013-1013Junctional diversity

36403519Number of V gene pairs

12(1)2Joints with N and P nucleotides

611355Joining Segments (J)

-often-rarelyD segments read in 3 frames

020~30Diversity Segment (D)

~70526951Variable Segment (V)αβλ + κH

© Current Biology / Garland

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-antibodies can recognize any chemical type of antigen. Recognition achieved through excellent shape complementarity.-~1000 to 1800Å2 of buried surface area, largely dominated by heavy chain (~2/3).-binding energetics primarily driven by desolvation, entropy driven, but also favorable enthalpy through extensive van der Waals contacts.

Large protein antigens interact with flat antibody binding sites through structurally discontinous “epitopes.”-what is an “epitope”?

•Smaller antigens such as peptides and haptensare usually buried in deep antibody binding sites.•Anti-peptide antibodies generally do not recognize peptide structures that are found in the folded protein.

-any part of a protein surface is antigenic. A protein can elicit antibodies to all exposed surfaces.-no correlation between a structural property of a protein and its antigenicity.

Three different mAbs to lysozyme

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-a single mAb can also recognize structurally different antigens.-case of anti-idiotypic antibody to a anti-lysozyme antibody.

mAb1

lysozyme

mAb1

mAb2

Antigen < Ab1 > Ab2

mutational effect of Alanine scan:

-energetic dissection of interface reveals that one mAb binds to twodifferent antigens with unique energetic landscape.-highlight the power of somatic mutation in affinity maturation.

Sundberg, Mariuzza et al.

-in general, affinity maturation improves complementarity with antigen

-but, somatic hypermutation can improve affinity through mutations at antibody sites not contacting antigen.

-30,000X higher affinity after nine replacement somatic mutations.- only 1 of 9 mutations contact antigen.

-germline antibodies appear to have flexible “plastic” combining sites and bind different haptens through induced-fit.-affinity maturation may freeze antibody binding site into optimal binding conformation, reducing the entropic cost of binding.

Bound(green/blue) versus free(grey) germline

Bound germline(green) versus free affinity-matured (NP-specific)

-affinity matured is specific for hapten 2, and has optimal CDR3 conformation frozen.

-germline Ab cross-reactswith two structurally differenthaptens.-each hapten induces uniqueCDR3 conformation

(Yin et al. J Mol Biol. 2003 Jul 18;330(4):651-6.)

Example:

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The MHC-fold

Class I Class II

T cell receptors are inherently cross-reactive

antibody/antigen TCR/peptide-MHC

ligand diversity

Affinity (K )

qualitatively similarbiological response

qualitatively differentbiological responses

mM

µM

mM

nM

D

-development / surveillance / alloreactivity -autoimmune reactions

-clearance

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The “docking” problem:How does TCR recognize peptide-MHC ?

1) “perpendicular” – Jorgensen et al., 1992- in class II IEk-MCC system used single-chain TCR-Fv transgenics to map peptide

contacts with CDR3 of TCR.- Found reverse charge-complementation in CDR3α with P3 peptide position.

2) “diagonal” - Sun et al., 1996.- in class I H-2Kb system, used mutants of Kb helices to profile reactivity of a

panel of Kb-restricted CTL’s. In general, mutants which disrupted reactivity were at thediagonal corners of MHC helices.

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•Vα lies over peptide N-terminal half.•CDR3s positioned over P4-P5 central peptideResidues.•Vβ lies over peptide C-terminal half.

Relatively (loosely) conserved TCR/pMHC docking orientation:

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Rudolph M et al. Curr. Opin. Imunol. 14, 52-65,2002

• “footprint” of TCR over pMHC reveals similar diagonal orientation in spite of NO common interatomic contacts.• what are the implications ?

•Claim was made that class II bound in a “perpendicular” orientation that was fundamentally different from class I (Reinherz et al., 2000).•However, CDR footprint is still diagonal, although body of TCR is orthogonal.

-In class II, peptide traverses entire length ofMHC groove, compared to short class I peptidesfixed at the ends.-TCR contact residues remain in similar positions.

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Possible structural rationale for TCR/MHC orientation: MHC “peaks.”

Table 2. Correlation of TCR Position and CD8 DependenceTCR/pMHC Group CD8-dependent? Docking AngleAHIII 12.2/p1049/A2 a No (Buslepp et al., 2003) 89°BM3.3/pBM1/Kb a No (Guimezanes et al., 2001) 58°LC13/FLRGRAYGL/B8 a No (Sewell et al, 1999) 57°JM22/MP/A2 a No (Lawson et al., 2001) 84°2C/SIYR/Kb b Yes (Daniels et al., 2000) 44°A6/Tax/A2 b Yes (data not shown) 58°B7/Tax/A2 b Yes (data not shown) 72°KB5-C20/pKB1/Kb b Yes (Guimezanes et al. 2001) 45°Docking angles are measured as the angle from the line formed by the amino and carboxyl ends of the class I MHC bound peptide.

What is diagonal orientation telling us ? What is its basis ?

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H-2D cells

H-2B mouse

2C CTL clone

Negative selection

H-2Kbm3H-2Ld

Positive selection

H-2Kb

Focus on a specific system: the 2C TCR.

TCR 2C Binding Specificity

Antagonist87KbRGYVYQELEVSV

Syngeneic

Allogeneic

Agonist (-ve selection)

56Kbm3EQYKFYSVdEV8

Agonist3.9LdQLSPFPFDLQL9

Weak agonist3.3LdLSPFFDLp2Ca

Very weak agonist120KbLSPFPFDLp2Ca

Weak agonist (positive selection)

74KbEQYKFYSVdEV8

ActivityKD (µM)MHC alleleSequencePeptide

SIYR SIYRYYGL Kb 54 Super-agonist

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TCR

MHC

EQYKFYSVTCR complexed with MHC presenting positively-selecting peptide:

TCR

MHC

Poor interface complementarity:

EQYKFYSV

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Model for thymic selection:

Ashton-Rickardt, others…

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Malissen et al. 2003

Allo-complexes show much greater interaction of TCR with Kb-bound peptide

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2C/H-2Kb/dEV8 2C/H-2Kb/SIYR

Degano et al., Immunity 2000 12, pp 251-61.

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Model of 2C/Ld-QL9 complex Structure of 2C/Kb-dEV8 complex

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Recommended references:

Antibody/antigenBraden BC, Goldman ER, Mariuzza RA, Poljak RJ.Anatomy of an antibody molecule: structure, kinetics, thermodynamics and mutational studies of the antilysozyme antibody D1.3.Immunol Rev. 1998 Jun; 163: 45-57

Davies DR, Cohen GH.Interactions of protein antigens with antibodies.Proc Natl Acad Sci U S A. 1996 Jan 9; 93(1): 7-12

Padlan EA.X-ray crystallography of antibodies.Adv Protein Chem. 1996; 49: 57-133.

Wilson IA, Stanfield RL.Antibody-antigen interactions: new structures and new conformational changes.Curr Opin Struct Biol. 1994 Dec; 4(6): 857-67

TCR/MHC

Garcia KC, Teyton L, Wilson IA.Structural basis of T cell recognition.Annu Rev Immunol. 1999; 17: 369-97

Housset D, Malissen B.What do TCR-pMHC crystal structures teach us about MHC restriction and alloreactivity?Trends Immunol. 2003 Aug; 24(8): 429-37. Review

Bankovich AJ, Garcia KC.Not just any T cell receptor will do.Immunity. 2003 Jan; 18(1): 7-11

Rudolph MG, Luz JG, Wilson IA.Structural and thermodynamic correlates of T cell signaling.Annu Rev Biophys Biomol Struct. 2002; 31: 121-49