Understanding Immune Recognition Understanding biology through structures Course

Understanding Immune Recognition Understanding biology through structures Course 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 structures Course work 2006

The Immunoglobin Fold Understanding biology through structures Course work 2006

Immunoglobin Fold • V and C domains share the basic Ig fold • Differences between the two domains • C domain is built of seven b-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 CDR 2 Understanding biology through structures Course work 2006

Structure of antibody Understanding biology through structures Course work 2006

Complementarity Determining Regions in Ig Understanding biology through structures Course work 2006

The Complementarity Determining Regions • Six loops of the VH (H 1, H 2 and H 3) and VL (L 1, L 2 and L 3) 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 • H 3 is the most variable of the loops and in all crystallographically solved antibody-antigen complexes makes several contacts with antigen Understanding biology through structures Course work 2006

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

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

Weak forces vs high affinity • 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 Understanding biology through structures Course work 2006

Antibody-Hapten Complex • 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 L 1 loops and a CDR H 3 loop with an "open" conformation, allowing the hapten to bind as much as 80% of its total surface in the interaction. Understanding biology through structures Course work 2006

Intimate interaction between Ab and Hapten Understanding biology through structures Course work 2006

Peptide Antibody Complex Understanding biology through structures Course work 2006

Protein Antibody Complex • In contrast, proteins preferentially to a relatively flat binding surface • In a "closed" CDR H 3 conformation, the CDR H 3 loop packs down onto the central cavity, and the protein antigen binds on top of it. Understanding biology through structures Course work 2006

Effector response is mediated via Ig-Fc. R 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 (CH) 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. • Fc. R recognize the Fc portion of antibodies not antigens Understanding biology through structures Course work 2006

The Fc-Fc Receptor complex • Fc. R plays important role in antibody mediated immune responses • Ig and Fc. R binding activates effector functions • Fc Receptor interacts with the CH 2 and CH 3 domains of Immunoglobulins Understanding biology through structures Course work 2006

Mode of interaction of Fc. R with difference Ig molecules Understanding biology through structures Course work 2006

Immune Recognition : MHC and TCR interactions Understanding biology through structures Course 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 structures Course 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 structures Course 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 structures Course 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 structures Course 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 Understanding biology through structures • MHC Class II – present exogenous antigen which has been phagocytosed and processed. eg. Bacteria – This is performed by professional antigen presenting cells eg macrophages Course 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 a-chain of 3 domains associated with b-2 microglobulin Understanding biology through structures • 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 Course work 2006

MOLECULES OF T LYMPHOCYTE RECOGNITION l. Major histocompatibility complex (MHC); human=Human Leukocyte Antigen (HLA); mouse=H-2 l. Gorer and Snell identified a genetic basis for graft rejection and Snell named it histocompatibility 2 (H-2). Nobel prize awarded to Snell. l. Highly polymorphic genes organized in a complex on chromosome 6 (human) and 17 (mouse). l. Glycoproteins expressed on the surface of cells. MHC class I is composed of one polypeptide, noncovalently associated with b 2 microglobulin. MHC class II is composed of two polypeptides, referred to as a and b. CD 8 T-CELL CD 4 T-CELL a b TCR ab CD 4 b 1 MHC CLASS II b CD 3 b 2 a 1 ANTIGEN PRESENTING CELL a b CD 8 15 aa peptide a 2 Understanding biology through structures a TCR ab a 2 MHC CLASS I CD 3 a 1 9 aa peptide b 2 m ANTIGEN PRESENTING CELL Course 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 Understanding biology through structures • 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 Course work 2006

MHC Class I and Class II Proteins Understanding biology through structures Course work 2006

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

Class I: Peptide Binding Understanding biology through structures Course work 2006

MHC-II Structure Understanding biology through structures Course work 2006

Peptide Binding by Major Histocompatibility Complex (MHC) Antigen-presenting Proteins MHC I • Peptides of intracellular origin • Peptides 9 -10 residues long • Deep pockets bind peptide sidechains • Deep pockets bind peptide Nand C-termini Understanding biology through structures MHC II • Peptides of extracellular origin • Peptides 15 residues or longer • Shallow pockets bind peptide sidechains • Peptide termini free • H-bonds to peptide backbone Course work 2006

Understanding biology through structures Course work 2006

MHC Polymorphism • 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 Understanding biology through structures Course work 2006

Location of Polymorphic Residues Understanding biology through structures Course work 2006

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

Understanding biology through structures Course work 2006

T-cell Receptor Understanding biology through structures Course 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 CD 4+ or CD 8+ fate. This activation involves other subunits of the receptor complex as well as other membraneassociated 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 structures Course work 2006

Understanding biology through structures Course work 2006

TCR binds peptide/MHC with a restricted (but variable) orientation Understanding biology through structures Course 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. 2003. Immunity 18, 7 -11 Understanding biology through structures Course work 2006

Understanding biology through structures Course work 2006

TRI-MOLECULAR COMPLEX CHARACTERISTICS -CDR 1 and CDR 2 interact with MHC molecules (a helices) -CDR 3 interacts with the peptide -interaction always in the same orientation -45 to 70 degrees angle related to peptide -Va see N-ter of the peptide -Vb see C-ter of the peptide Understanding biology through structures Course 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 structures Course work 2006

Understanding biology through structures Course work 2006

Recognition of the Super Antigens Understanding biology through structures Course work 2006

Antigen Recognition by Antibodies (Ab) and T-cell Receptors (TCR) Ab - Ag • Surface area ~ 2 x 750 Å2 • Epitope discontinuous in antigen (Ag) sequence Understanding biology through structures TCR – MHC/peptide • Surface area ~ 2 x 1000 Å2 • Ag peptide contributes only 40% of surface area • Epitope continuous in Ag sequence • Otherwise similar to Ab Ag recognition Course work 2006

PARADOX -TCR-MHC interaction has a weak affinity -affinity ~ 10 m. M -half-life ~10 s -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 (>2 h) Understanding biology through structures Course work 2006