microbilogy smc7

8/11/2019 microbilogy smc7

1/53

Understanding biology through structures Course work 2006

Understanding Immune Recognition


2/53


Antigen Recognition B cells can recognise antigens via their surface Ig molecules

T cells can only recognise antigen in association with a MajorHistocompatibility Complex (MHC) molecule.


3/53


Antigen Recognition

B cells can recognise antigens via theirsurface Ig molecules

T cells can only recognise antigen inassociation with a Major HistocompatibilityComplex (MHC) molecule.


4/53


The Immunoglobin Fold


5/53


Immunoglobin Fold

V and C domains share the basic Ig fold

Differences between the two domains

C domain is built of sevenb-strandsarranged so that four strands form onesheet and three strands form a secondsheet.

The strands are closely packed together andjoined by a single disulphide bond

Most of the invariant residues of theconstant domain are in the sheets

Overall structure of the V domain verysimilar but there are nine strands instead ofseven. The two additional strands harbourCDR2


6/53


Structure of antibody


7/53Understanding biology through structures Course work 2006

Complementarity Determining Regions in Ig



Six loops of the VH (H1, H2 and H3) and VL (L1, L2 and L3) domains create agreat variety of surfaces

Deep binding cavities: such as those seen in some antibody-hapten complexes

Wide pockets : seen in certain antibody-peptide complexes

Flat surfaces : seen in antibody-protein interactions

H3 is the most variable of the loops and in all crystallographically solvedantibody-antigen complexes makes several contacts with antigen

The Complementarity Determining Regions



What Do Antibodies Recognize?

1. Proteins (conformational determinants, denatured or proteolyzeddeterminants)

2. Nucleic acids3. Polysaccharides4. Some lipids5. Small chemicals (haptens)



Antibodies bind to antigens by

recognizing a large surface, andthrough surface complementarity.

Thus, these complexes have a veryhigh affinity for each other.

Antigen:Antibody complex



The interaction between an antigen and antibody can be very strong, and yet allof the forces involved are considered to be relatively weak. How can weakhydrogen bonds, electrostatic attractions, hydrophobic forces, and van der Waalscontacts lead to a high affinity?

Contact between antigen and antibody occurs over a wide surface area, allowingmultiple weak interactions that give a strong affinity

Hydrogen bonds join the antibody and antigen over a wide surface area. Other weak forces, including van der Waals forces, electrostatic attractions andhydrophobic forces, add to the strength and specificity of antibody/antigenbinding

Weak forces vs high affinity



Haptens, having a limited total surface area, deeply embed themselves into the VL/VHdimer interface

Hapten binding antibodies frequently show a deep central cavity, long CDR L1 loops aCDR H3 loop with an "open" conformation, allowing the hapten to bind as much as 80of its total surface in the interaction.

Antibody-Hapten Complex



Intimate interaction between Ab and Hapten



Peptide Antibody Complex



In contrast, proteins preferentially to a relatively flat binding surface

In a "closed" CDR H3 conformation, the CDR H3 loop packs down onto thecentral cavity, and the protein antigen binds on top of it.

Protein Antibody Complex



Effector response is mediated via Ig-FcRcomplex formation

Antibodies not only must recognize antigen, but also musinvoke responses effector functions that will remove theantigen and kill the pathogen.

Variable regions of antibody are the sole agents of binding tantigen. The heavy chain constant region (CH ) is responsible for

interactions with other proteins (e.g. complement), cell

(elements of innate immune system), and tissues that result in teffector functions of the humoral response. FcR recognize the Fc portion of antibodies not antigens



The Fc-Fc Receptor complex FcR plays important role in antibody mediated immune responses

Ig and FcR binding activates effector functions

Fc Receptor interacts with the CH2 and CH3 domains of Immunoglobulins



Mode ofinteraction ofFcR withdifference Igmolecules


19/53



Antigen Recognition

B cells can recognise antigens via theirsurface Ig molecules

T cells can only recognise antigen inassociation with a Major HistocompatibilityComplex (MHC) molecule.



T cells

T cells display TCR as their antigen recognition protein

When stimulated they become Cytotoxic or Helper T cells

Secrete cytokines that recruit other cells of the IS

TCRs only recognise short peptides.



MHC & T cells T cells have a requirement to recognise both the ANTIGEN and the

MHC molecule. This is because the molecular structure of the MHC- Antigen complex is arranged so that some of the polymorphic aminoacids of the MHC molecule are in direct contact with the TCR Therefore T cell recognition of antigen is said to be MHC restricted.


23/53


Antigen Processing andPresentation Fragmentation of protein into peptides

Association of peptide with an MHC molecule Transport to cell surface for expression Different cellular pathways for association of peptide with MHC class I

and class II molecules


24/53


MHC & Antigens

MHC Class I present endogenously

derived peptides. these can be either self or

derived from viruses because MHC Class I is

present on all cells any cellcan interact with T cells if

infected by a virus

MHC Class II present exogenous

antigen which has beenphagocytosed and

processed.eg. Bacteria This is performed byprofessional antigenpresenting cells egmacrophages


25/53


MHC MHC Class I

detected on allnucleated cells

very highly polymorphic Tight fit for peptides of

only about 9 aa consists of ana -chain

of 3 domains associated withb-2 microglobulin

MHC Class II seen only on the

professional antigenprocessing cells e.g

macrophage slightly less

polymorphic accepts peptides of up

to 15 aa acids


26/53


b

CD4 T-CELL

TCR bCD4

CD3

9 aapeptide15 aa

peptide

CD8 T-CELL

CD8

b

TCR b

CD3

ANTIGEN PRESENTING CELL

MHCCLASS II

1 1

b2 2

Major histocompatibility complex (MHC); human=Human Leukocyte Antigen (HLA); mouse=H-2Gorer and Snell identified a genetic basis for graft rejection and Snell named it histocompatibility 2

(H-2). Nobel prize awarded to Snell.

Highly polymorphic genes organized in a complex on chromosome 6 (human) and 17 (mouse).Glycoproteins expressed on the surface of cells. MHC class I is composed of one polypeptide, non-

covalently associated with b2microglobulin. MHC class II is composed of two polypeptides, referredto as and b .

MOLECULES OF T LYMPHOCYTE RECOGNITION

CLASS IMHC b 2m

2

1

3

ANTIGEN PRESENTING CELL

MHC Cl I d Cl II P i


27/53


MHC Class I and Class II Proteins Class I

Alpha Chain 3 External domains 1 Transmembrane 1 Cytoplasmic tail Encoded in MHC

Beta-2 Microglobulin 1 External domain

No transmembrane No Cytoplasmic tail Not encoded in MHC

Class II Alpha Chain

2 External domains 1 Transmembrane 1 Cytoplasmic Tail Encoded in MHC

Beta Chain 2 External domains

1 Transmembrane 1 Cytoplasmic Tail Encoded in MHC

MHC Cl I d Cl II P i


28/53


MHC Class I and Class II Proteins


29/53


Peptides bind to MHC molecules in a polyproline II conformation


30/53


Class I:Peptide Binding


31/53


MHC-II Structure


32/53


Peptide Binding by Major Histocompatibility Complex (MHC) Antigen-presenting Proteins

Peptides of intracellular origin Peptides 9-10 residues long Deep pockets bind peptide

sidechains Deep pockets bind peptide N-

and C-termini

Peptides of extracellular origin Peptides 15 residues or longer Shallow pockets bind peptide

sidechains Peptide termini free H-bonds to peptide backbone

MHC I MHC II


33/53



34/53


Both Class I and Class II genes are highlypolymorphic

Most polymorphic residues of Class I are inthe alpha 1 and alpha 2 domains

Most polymorphic residues of Class II are inthe alpha 1 and beta 1 domains

MHC Polymorphism


35/53


36/53


Location of Polymorphic Residues


37/53


Allelic variation in MHC occurs at the peptide binding site and on the top/sidesof the binding cleft


38/53



39/53


40/53


The T cell receptor (TCR) is a complex of integral membrane proteins that participates in the activation of T cells in response to the presentation of antigen.Specific recognition and binding by the clonotype-specific a/b heterodimer leads toactivation of transcription and commitment of the T cell to CD4+ or CD8+ fate. Thisactivation involves other subunits of the receptor complex as well as other membrane-associated molecules that couple the extracellular liganding event to downstreamsignaling pathways such as protein phosphorylation, the release of inositol phosphatesand the elevation of intracellular calcium levels.


41/53



42/53



43/53


b d d h


44/53


TCR binds peptide/MHC with arestricted (but variable) orientation


45/53


peptide binding interface: 21-34% proportion of TCR contacts with the peptide:26-47%contact are different between TCR-MHC complex

-the contribution to the binding energy is still uncleared!

Bandovich and Garcia. 2003. Immunity 18,7-11


46/53



47/53


48/53


TRI-MOLECULAR COMPLEXCHARACTERISTICS

- most of the binding interface is between the TCRand MHC helices

- conformational change in the TCR CDR loopsenhances TCR crossreactivity

- no conformational change in the TCR constant region(except in one complex out of ten)


49/53



50/53



51/53


Recognition of the Super Antigens


52/53


Antigen Recognition by Antibodies (Ab)

and T-cell Receptors (TCR)

Surface area ~ 2x750 2

Epitope discontinuous inantigen (Ag) sequence

Surface area ~ 2x1000 2

Ag peptide contributesonly 40% of surface area

Epitope continuous in Agsequence

Otherwise similar to Ab - Ag recognition

Ab - Ag TCR MHC/peptide


53/53

PARADOX

-TCR-MHC interaction has a weak affinity-affinity ~ 10 mM-half-life ~10s

-restricted numbers of ligands (~100) are displayed at the surfaceof antigen presenting cells

-T cell activation requires a long interaction with antigen presentingcells (>2h)

microbilogy smc7

Documents