Antigen Recognition by B-cell and T-cell Receptors

Structure of the Antibody / Immunoglobulin Molecule

• Basic Heterotetrameric Structure:

  • Immunoglobulins (antibodies) are Y-shaped glycoproteins constructed from four polypeptide chains: two identical heavy chains (H chains, $\approx 50\,\text{kDa}$ each) and two identical light chains (L chains, $\approx 25\,\text{kDa}$ each), giving a total molecular weight of approximately $150\,\text{kDa}$.

  • The chains are linked covalently by interchain disulfide bonds and noncovalently through hydrophobic and electrostatic interactions.

• Variable and Constant Regions:

  • Variable ($V$) Region: Located at the amino-terminal ($\text{N}$ terminus) of both heavy ($V_H$) and light ($V_L$) chains. The paired $V_H$ and $V_L$ domains form two identical antigen-binding sites at the tips of the Y-arms.

  • Constant ($C$) Region: Located at the carboxyl-terminal ($\text{C}$ terminus) of both heavy ($C_H1, C_H2, C_H3$) and light ($C_L$) chains. The constant region engages host effector mechanisms (e.g., complement activation, Fc receptor binding).

• Domain Layout:

  • Light Chain Domains: Comprises one variable domain ($V_L$) and one constant domain ($C_L$).

  • Heavy Chain Domains: Comprises one variable domain ($V_H$) and three or four constant domains ($C_H1, C_H2, C_H3$, and in some classes $C_H4$).

  • Carbohydrate Attachment: A conserved $N$-linked carbohydrate moiety is attached to the $C_H2$ domain of each heavy chain, maintaining structural stability and mediating Fc receptor interactions.

  • Hinge Region: A flexible segment rich in proline and cysteine residues situated between the $C_H1$ and $C_H2$ domains of the heavy chain, housing the interchain disulfide bonds that link the heavy chains.

Proteolytic Cleavage and Structural Flexibility of Immunoglobulins

• Proteolytic Cleavage by Papain:

  • Cleaves the heavy chain polypeptide backbone on the amino-terminal side of the interchain hinge disulfide bonds.

  • Yields three separate fragments:

  • Two identical $Fab$ (Fragment antigen-binding) fragments: Monovalent fragments consisting of a complete light chain ($V_L + C_L$) paired with the $V_H$ and $C_H1$ domains of a heavy chain. Each $Fab$ fragment retains a single, functional antigen-binding site.

  • One $Fc$ (Fragment crystallizable) fragment: Composed of the paired $C_H2$ and $C_H3$ domains (carboxyl-terminal halves of both heavy chains) linked by disulfide bonds. It readily crystallizes out of solution and interacts with effector molecules.

• Proteolytic Cleavage by Pepsin:

  • Cleaves the heavy chain polypeptide backbone on the carboxy-terminal side of the hinge disulfide bonds.

  • Yields a single bivalent antigen-binding fragment designated $F(ab')_2$, consisting of both $Fab$-like arms connected by the intact hinge disulfide bridge.

  • The remainder of the heavy chain constant region is degraded into multiple smaller fragments, the largest designated $pFc'$.

• Hinge Region Flexibility:

  • The hinge region acts as a flexible joint that allows relative movement and rotation between the two $Fab$ arms and the $Fc$ stem.

  • Segmental flexibility permits the angle between the two $Fab$ arms to vary widely (e.g., from $60^\text{o}$ to $90^\text{o}$ up to $180^\text{o}$).

  • Electron micrographs at high magnification ($\times 300{,}000$) show that antibodies binding small multivalent hapten molecules form dynamic geometric immune complexes (e.g., triangular complexes at $60^\text{o}$ inter-arm angles and square complexes at $90^\text{o}$ inter-arm angles).

  • This structural adaptability allows bivalent antibodies to engage epitopes presented at varying distances and spatial orientations on pathogen surfaces.

Domain Folding and Hypervariable Regions of Immunoglobulins

• The Immunoglobulin Fold:

  • Both $V$ and $C$ domains share a characteristic tertiary fold consisting of a structural sandwich formed by two antiparallel $\beta$-sheets linked by a central conserved intrachain disulfide bond between cysteine residues.

  • Constant Domain ($C_L$ / $C_H$): Composed of 7 antiparallel $\beta$-strands arranged into two sheets: a 3-stranded sheet ($C, F, G$) and a 4-stranded sheet ($D, E, B, A$).

  • Variable Domain ($V_L$ / $V_H$): Contains 9 antiparallel $\beta$-strands arranged in two sheets ($D, E, B, A$ and $G, F, C, C', C''$). The two additional strands ($C'$ and $C''$) create a larger domain with extended loops at the amino-terminal tip.

• Hypervariable Regions (CDRs) and Framework Regions:

  • Sequence variability analysis across variable domains reveals four conserved Framework Regions ($FR1, FR2, FR3, FR4$) that fold into the core $\beta$-sheet structural scaffold.

  • Interspersed between the framework regions are three Hypervariable (HV) regions, also known as Complementarity-Determining Regions ($CDR1, CDR2, CDR3$).

  • Residue positions in Light Chain $V$ Region:

  • $FR1$: Residues $1$–$23$

  • $HV1$ ($CDR1$): Residues $24$–$34$

  • $FR2$: Residues $35$–$49$

  • $HV2$ ($CDR2$): Residues $50$–$56$

  • $FR3$: Residues $57$–$88$

  • $HV3$ ($CDR3$): Residues $89$–$97$

  • $FR4$: Residues $98$–$107$

  • Similar hypervariable peaks exist in the Heavy Chain $V$ region at corresponding positions ($HV1, HV2, HV3$).

• Spatial Organization of the Antigen-Binding Site:

  • When the variable domain folds, the $\beta$-strands form the scaffold, projecting the flexible loops corresponding to $HV1, HV2$, and $HV3$ to the exterior at the top of the domain.

  • Pairing of $V_H$ and $V_L$ domains brings the 3 hypervariable loops of $V_L$ and the 3 hypervariable loops of $V_H$ together, creating a single contiguous 6-loop surface that constitutes the antigen-binding site (paratope).

Structural Basis of Antigen Binding and Noncovalent Interactions

• Topography of Antigen-Binding Sites:

  • Pocket Binding: A deep central pocket or cavity formed between $V_H$ and $V_L$ domains accommodates small, compact molecules or haptens (e.g., phosphorylcholine, dinitrophenol).

  • Groove Binding: An elongated, extended cleft formed by CDR loops accommodates linear polymeric structures such as peptides, oligosaccharides, or nucleic acids.

  • Extended Planar Surface Binding: A broad, flat contact interface spanning all six CDR loops engages large native macromolecular antigens (e.g., globular proteins like hen egg-white lysozyme or viral surface capsids).

  • Protruding Surface / Knob Binding: Extended CDR loops project outward to penetrate deep active-site pockets or clefts on target enzyme antigens.

• Noncovalent Forces Driving Binding:

  • Antigen-antibody interaction is strictly noncovalent and reversible, depending on fine structural complementarity.

  • Electrostatic Forces: Ionic attraction between oppositely charged side chains (e.g., basic amino acid $\text{-NH}_3^+$ and acidic amino acid $\text{-OOC-}$).

  • Hydrogen Bonds: Dipole-dipole interactions where a hydrogen atom with a partial positive charge ($\delta^+$) is shared between electronegative donor and acceptor atoms ($\text{N}, ext{O}$) carrying partial negative charges ($\delta^-$).

  • Van der Waals Forces: Transient fluctuations in electron density around molecules create temporary dipoles that induce complementary polarization in neighboring atoms.

  • Hydrophobic Forces: Entropically driven aggregation of nonpolar residue side chains to exclude water molecules from the contact interface, stabilized by secondary van der Waals contacts.

Structure and Comparison of the T-cell Receptor (TCR)

• Architecture of the $\alpha:\beta$ T-Cell Receptor:

  • The $\alpha:\beta$ T-cell receptor (TCR) is a membrane-bound heterodimeric glycoprotein composed of one $\alpha$ chain ($\approx 45\text{--}50\,\text{kDa}$) and one $\beta$ chain ($\approx 40\text{--}45\,\text{kDa}$) joined by an extracellular interchain disulfide bond.

  • Each polypeptide chain consists of two Ig-like domains: an amino-terminal Variable domain (V__\beta, V__\beta) and a membrane-adjacent Constant domain (C__\beta, C__\beta).

  • Stalk Segment: Flexible extracellular region containing the interchain disulfide bridge.

  • Transmembrane Region: Contains positively charged amino acid residues ($+$, e.g., Lysine, Arginine) that mediate association with negatively charged residues in the CD3 signaling complex ($\gamma, \delta, \epsilon$, and $\zeta$ chains).

  • Cytoplasmic Tail: Short carboxy-terminal intracellular segment lacking intrinsic enzymatic activity.

• Structural Comparison with Immunoglobulins:

  • Valency: TCRs are monovalent with a single antigen-binding site ($V_\alpha + V_\beta$), whereas antibodies are bivalent (two Fab arms).

  • Solubility: TCRs function strictly as cell-surface membrane receptors; antibodies function as both membrane receptors and secreted soluble proteins.

  • Antigen Nature: Antibodies recognize native, three-dimensional conformational structures (proteins, glycans, lipids); TCRs recognize short linear peptides presented on self Major Histocompatibility Complex (MHC) molecules.

  • Hypervariable Loops: TCR variable domains contain loops analogous to antibody CDRs ($CDR1, CDR2, CDR3$). TCR$\beta$ possesses an additional hypervariable loop, $HV4$, which does not contact the bound peptide but interacts with superantigens.

Structural Comparison of MHC Class I and MHC Class II Molecules

• MHC Class I Molecular Architecture:

  • Heterodimer consisting of a heavy transmembrane $\alpha$ chain ($\approx 44\,\text{kDa}$) noncovalently associated with a non-polymorphic, soluble light chain, $\beta_2\text{-microglobulin}$ ($\beta_2\text{m}$, $\approx 12\,\text{kDa}$).

  • The heavy $\alpha$ chain folds into three domains: $\alpha_1, \alpha_2$, and $\alpha_3$.

  • Peptide-Binding Cleft: Formed by the $\alpha_1$ and $\alpha_2$ domains; consists of two parallel $\alpha$-helical walls supported by a platform floor of 8 antiparallel $\beta$-strands.

  • Membrane Anchor: Transmembrane region and cytoplasmic tail are located exclusively on the $\alpha_3$ domain of the heavy chain. $\beta_2\text{m}$ has no transmembrane domain and does not anchor directly in the cell membrane.

• MHC Class II Molecular Architecture:

  • Heterodimer consisting of two noncovalently linked, membrane-spanning polypeptide chains: an $\alpha$ chain ($\approx 33\,\text{kDa}$) and a $\beta$ chain ($\approx 28\,\text{kDa}$).

  • Domain organization: $\alpha_1$ and $\alpha_2$ on the $\alpha$ chain; $\beta_1$ and $\beta_2$ on the $\beta$ chain.

  • Peptide-Binding Cleft: Formed jointly by the $\alpha_1$ domain of the $\alpha$ chain and the $\beta_1$ domain of the $\beta$ chain (each supplying one $\alpha$-helix and half of the $\beta$-sheet floor).

  • Membrane Anchors: Both $\alpha$ and $\beta$ chains possess individual transmembrane domains and cytoplasmic tails.

Peptide Binding Mechanisms by MHC Class I and Class II Molecules

• MHC Class I Peptide Binding Features:

  • Accommodates short peptides, typically 8 to 10 amino acid residues in length (optimally 9 residues).

  • The peptide-binding cleft ends are blocked by conserved amino acid side chains.

  • Peptide termini are locked into deep pockets at both ends of the cleft: Free $N$-terminal ($\text{-NH}_3^+$) and $C$-terminal ($\text{-COO}^-$) groups form extensive hydrogen bonds with invariant MHC residues.

  • Anchor Residues: Peptides feature conserved anchor amino acids at specific positions (typically position 2, P2, and the $C$-terminus, P9) that insert hydrophobic or charged side chains into deep complementary pockets on the cleft floor (e.g., Leucine L, Isoleucine I, Valine V, Tyrosine Y).

• MHC Class II Peptide Binding Features:

  • Accommodates longer peptides, typically 13 to 17+ amino acid residues in length.

  • The peptide-binding cleft ends are open, allowing the ends of the bound peptide to extend beyond the cleft boundaries.

  • The peptide adopts an extended polyproline II helical conformation at a uniform height along the floor of the groove.

  • Binding stability relies on hydrogen bonds formed along the peptide main-chain backbone and conserved anchor residues distributed along the sequence (e.g., positions P1, P4, P6, P9).

T-cell Receptor Interaction with MHC:Peptide Complexes

• Orientation and Geometry of Engagement:

  • The TCR binds diagonally across the top of the MHC:peptide complex.

  • $V_\alpha$ domain positioned over the amino-terminal half of the bound peptide and the $\alpha_2$ helix of MHC class I (or $\beta_1$ helix of MHC class II).

  • $V_\beta$ domain positioned over the carboxy-terminal half of the bound peptide and the $\alpha_1$ helix of MHC class I (or $\alpha_1$ helix of MHC class II).

• Functional Role of CDR Loops in Dual Recognition:

  • Germline-encoded $CDR1$ and $CDR2$ loops (from both $V_\alpha$ and $V_\beta$) make primary contacts with the conserved $\alpha$-helical walls of the MHC molecule.

  • Hypervariable $CDR3$ loops ($3\alpha$ and $3\beta$) reside centrally directly above the peptide-binding cleft, making primary contacts with side chains of the bound peptide (specifically central residues at positions P5 through P8).

  • Dual recognition ensures both self-MHC restriction (via $CDR1/CDR2$) and antigen specificity (via $CDR3$).

Structures and Functions of CD4 and CD8 Coreceptors

• CD4 Coreceptor Structure and Specificity:

  • Single-chain monomeric transmembrane glycoprotein containing 4 extracellular Ig-like domains ($D_1, D_2, D_3, D_4$) arranged in a rigid rod connected by a flexible hinge.

  • Selectively binds conserved, non-polymorphic regions on the $\beta_2$ domain of MHC class II molecules away from the peptide cleft.

  • Expressed on helper T cells ($\text{CD4}^+$ T cells); recruits Lck tyrosine kinase to the TCR signaling complex upon engagement.

• CD8 Coreceptor Structure and Specificity:

  • Membrane-bound heterodimer composed of an $\alpha$ chain and a $\beta$ chain (or homodimer $\alpha\alpha$) joined by interchain disulfide bonds.

  • Each chain contains a single extracellular Ig-like domain connected to the membrane anchor by an extended, heavily O-glycosylated polypeptide stalk.

  • Selectively binds conserved, non-polymorphic regions on the $\alpha_3$ domain of MHC class I molecules.

  • Expressed on cytotoxic T cells ($\text{CD8}^+$ T cells); enhances sensitivity to presented foreign peptides by stabilizing TCR-MHC class I interactions.

Tissue Distribution of MHC Class I and Class II Expression

• MHC Class I vs Class II Expression Profile:

  • MHC Class I: Expressed broadly across virtually all nucleated cells, enabling surveillance by $\text{CD8}^+$ cytotoxic T cells.

  • MHC Class II: Restricted primarily to professional antigen-presenting cells (APCs) such as dendritic cells, B cells, macrophages, and thymic epithelial cells, for presentation to $\text{CD4}^+$ helper T cells.

• Detailed Tissue Expression Table:

  • Lymphoid Tissues:

  • T cells: MHC Class I ($+++$), MHC Class II ($+^*$ on activated human T cells, resting T cells are negative).

  • B cells: MHC Class I ($+++$), MHC Class II ($+++$).

  • Macrophages: MHC Class I ($+++$), MHC Class II ($++$).

  • Dendritic cells: MHC Class I ($+++$), MHC Class II ($+++$).

  • Thymic epithelial cells: MHC Class I ($+$), MHC Class II ($+++$).

  • Other Nucleated Cells:

  • Neutrophils: MHC Class I ($+++$), MHC Class II (-$).\n * **Hepatocytes:** MHC Class I (+$), MHC Class II ($-$).

  • Kidney cells: MHC Class I ($+$), MHC Class II (-$).\n * **Brain cells:** MHC Class I (+$), MHC Class II ($-^\dagger; inducible on microglial cells upon cytokine activation).\n * **Nonnucleated Cells:**\n * **Red Blood Cells (Erythrocytes):** MHC Class I (-$), MHC Class II (-$).\n\n\n\n# Structure and Properties of \gamma:\delta T-cell Receptors\n\n* **Architecture of \gamma:\delta T-Cell Receptors:**\n * Alternative heterodimeric receptor composed of a \gamma$chain and a$\delta$chain, each containing variable ($V_\gamma, V_\delta$) and constant ($C_\gamma, C_\delta) domains.\n * Shares basic domain structure and Ig-fold geometry with \alpha:\beta$$ TCRs, but exhibits different binding site topography and conformational mobility.

• Antigen Recognition Characteristics:

  • Recognition is non-MHC restricted: Does not require classical MHC class I or class II antigen presentation.

  • Capable of directly binding intact proteins, non-classical MHC molecules (e.g., CD1, MICA/MICB), and small non-peptide phosphorylated metabolites (phosphoantigens).