Understanding biology through structuresCourse work 2006 Understanding Immune Recognition

Understanding biology through structuresCourse work 2006 Antigen Recognition B cells can recognise antigens via their surface Ig molecules T cells can only recognise antigen in association with a Major Histocompatibility Complex (MHC) molecule.

Understanding biology through structuresCourse work 2006 Antigen Recognition B cells can recognise antigens via their surface Ig molecules T cells can only recognise antigen in association with a Major Histocompatibility Complex (MHC) molecule.

Understanding biology through structuresCourse work 2006 The Immunoglobin Fold

Understanding biology through structuresCourse work 2006 Immunoglobin Fold V and C domains share the basic Ig fold Differences between the two domains C domain is built of seven  -strands arranged so that four strands form one sheet and three strands form a second sheet. The strands are closely packed together and joined by a single disulphide bond Most of the invariant residues of the constant domain are in the sheets Overall structure of the V domain very similar but there are nine strands instead of seven. The two additional strands harbour CDR2

Understanding biology through structuresCourse work 2006 Structure of antibody

Understanding biology through structuresCourse work 2006 Complementarity Determining Regions in Ig

Understanding biology through structuresCourse work 2006 Six loops of the VH (H1, H2 and H3) and VL (L1, L2 and L3) domains create a great 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 solved antibody-antigen complexes makes several contacts with antigen The Complementarity Determining Regions

Understanding biology through structuresCourse work 2006 What Do Antibodies Recognize? 1.Proteins (conformational determinants, denatured or proteolyzed determinants) 2.Nucleic acids 3.Polysaccharides 4.Some lipids 5.Small chemicals (haptens)

Understanding biology through structuresCourse work 2006 Antibodies bind to antigens by recognizing a large surface, and through surface complementarity. Thus, these complexes have a very high affinity for each other. Antigen:Antibody complex

Understanding biology through structuresCourse work 2006 The interaction between an antigen and antibody can be very strong, and yet all of the forces involved are considered to be relatively weak. How can weak hydrogen bonds, electrostatic attractions, hydrophobic forces, and van der Waals contacts lead to a high affinity? Contact between antigen and antibody occurs over a wide surface area, allowing multiple 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 and hydrophobic forces, add to the strength and specificity of antibody/antigen binding Weak forces vs high affinity

Understanding biology through structuresCourse work 2006 Haptens, having a limited total surface area, deeply embed themselves into the VL/VH dimer interface Hapten binding antibodies frequently show a deep central cavity, long CDR L1 loops and a CDR H3 loop with an "open" conformation, allowing the hapten to bind as much as 80% of its total surface in the interaction. Antibody-Hapten Complex

Understanding biology through structuresCourse work 2006 Intimate interaction between Ab and Hapten

Understanding biology through structuresCourse work 2006 Peptide Antibody Complex

Understanding biology through structuresCourse work 2006 In contrast, proteins preferentially to a relatively flat binding surface In a "closed" CDR H3 conformation, the CDR H3 loop packs down onto the central cavity, and the protein antigen binds on top of it. Protein Antibody Complex

Understanding biology through structuresCourse work 2006 Effector response is mediated via Ig-FcR complex formation Antibodies not only must recognize antigen, but also must invoke responses – effector functions – that will remove the antigen and kill the pathogen. Variable regions of antibody are the sole agents of binding to antigen. The heavy chain constant region (C H ) is responsible for interactions with other proteins (e.g. complement), cells (elements of innate immune system), and tissues that result in the effector functions of the humoral response. FcR recognize the Fc portion of antibodies not antigens

Understanding biology through structuresCourse work 2006 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

Understanding biology through structuresCourse work 2006 Mode of interaction of FcR with difference Ig molecules

Understanding biology through structuresCourse work 2006 Immune Recognition : MHC and TCR interactions

Understanding biology through structuresCourse work 2006 Antigen Recognition B cells can recognise antigens via their surface Ig molecules T cells can only recognise antigen in association with a Major Histocompatibility Complex (MHC) molecule.

Understanding biology through structuresCourse work 2006 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 TCR’s only recognise short peptides.

Understanding biology through structuresCourse work 2006 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 amino acids of the MHC molecule are in direct contact with the TCR –Therefore T cell recognition of antigen is said to be MHC ‘restricted’.

Understanding biology through structuresCourse work 2006 Antigen Processing and Presentation 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

Understanding biology through structuresCourse work 2006 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 cell can interact with T cells if infected by a virus MHC Class II –present exogenous antigen which has been phagocytosed and processed.eg. Bacteria –This is performed by professional antigen presenting cells eg macrophages

Understanding biology through structuresCourse work 2006 MHC MHC Class I –detected on all nucleated cells –very highly polymorphic –Tight fit for peptides of only about 9 aa –consists of an  -chain of 3 domains associated with  -2 microglobulin MHC Class II –seen only on the ‘professional antigen processing cells’ e.g macrophage –slightly less polymorphic –accepts peptides of up to 15 aa acids

Understanding biology through structuresCourse work 2006   CD4 T-CELL TCR  CD4 CD3 9 aa peptide 15 aa peptide CD8 T-CELL CD8   TCR  CD3   ANTIGEN PRESENTING CELL MHC CLASS II 11 11 22 22 Major histocompatibility complex (MHC); human=Human Leukocyte Antigen (HLA); mouse=H-2 Gorer 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  2microglobulin. MHC class II is composed of two polypeptides, referred to as  and . MOLECULES OF T LYMPHOCYTE RECOGNITION CLASS I MHC 2m2m 22 11 33 ANTIGEN PRESENTING CELL

Understanding biology through structuresCourse work 2006 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

Understanding biology through structuresCourse work 2006 MHC Class I and Class II Proteins

Understanding biology through structuresCourse work 2006 Peptides bind to MHC molecules in a polyproline II conformation

Understanding biology through structuresCourse work 2006 Class I:Peptide Binding

Understanding biology through structuresCourse work 2006 MHC-II Structure

Understanding biology through structuresCourse work 2006 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 IMHC II

Understanding biology through structuresCourse work 2006

Both Class I and Class II genes are highly polymorphic Most polymorphic residues of Class I are in the alpha 1 and alpha 2 domains Most polymorphic residues of Class II are in the alpha 1 and beta 1 domains MHC Polymorphism

Understanding biology through structuresCourse work 2006 Location of Polymorphic Residues

Understanding biology through structuresCourse work 2006 Location of Polymorphic Residues

Understanding biology through structuresCourse work 2006 Allelic variation in MHC occurs at the peptide binding site and on the top/sides of the binding cleft

Understanding biology through structuresCourse work 2006

T-cell Receptor

Understanding biology through structuresCourse work 2006 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 to activation of transcription and commitment of the T cell to CD4+ or CD8+ fate. This activation involves other subunits of the receptor complex as well as other membrane- associated molecules that couple the extracellular liganding event to downstream signaling pathways such as protein phosphorylation, the release of inositol phosphates and the elevation of intracellular calcium levels.

Understanding biology through structuresCourse work 2006

TCR binds peptide/MHC with a restricted (but variable) orientation

Understanding biology through structuresCourse work 2006 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 Immunity 18,7-11

Understanding biology through structuresCourse work 2006

-CDR1 and CDR2 interact with MHC molecules (  helices) -CDR3 interacts with the peptide -interaction always in the same orientation -45 to 70 degrees angle related to peptide -V  see N-ter of the peptide -V  see C-ter of the peptide TRI-MOLECULAR COMPLEX CHARACTERISTICS

Understanding biology through structuresCourse work 2006 TRI-MOLECULAR COMPLEX CHARACTERISTICS - most of the binding interface is between the TCR and MHC helices - conformational change in the TCR CDR loops enhances TCR crossreactivity - no conformational change in the TCR constant region (except in one complex out of ten)

Understanding biology through structuresCourse work 2006

Recognition of the Super Antigens

Understanding biology through structuresCourse work 2006 Antigen Recognition by Antibodies (Ab) and T-cell Receptors (TCR) Surface area ~ 2x750 Å 2 Epitope discontinuous in antigen (Ag) sequence Surface area ~ 2x1000 Å 2 Ag peptide contributes only 40% of surface area Epitope continuous in Ag sequence Otherwise similar to Ab - Ag recognition Ab - AgTCR – MHC/peptide

Understanding biology through structuresCourse work 2006 PARADOX -TCR-MHC interaction has a weak affinity -affinity ~ 10  M -half-life ~10s -restricted numbers of ligands (~100) are displayed at the surface of antigen presenting cells -T cell activation requires a long interaction with antigen presenting cells (>2h)