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Immunology_Janeway. Dellinger, R.P.: Inflammation and coagulation: implications for the septic patient. Clin. Infect. Dis. 2003, 36:1259–1265. Georgel, P., Naitza, S., Kappler, C., Ferrandon, D., Zachary, D., Swimmer, C., Kopczynski, C., Duyk, G., Reichhart, J.M., and Hoffmann, J.A.: Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis. Dev. Cell 2001, 1:503–514. Pfeffer, K.: Biological functions of tumor necrosis factor cytokines and their receptors. Cytokine Growth Factor Rev. 2003, 14:185–191. Rutschmann, S., Jung, A.C., Zhou, R., Silverman, N., Hoffmann, J.A., and Ferrandon, D.: Role of Drosophila IKKγ in a toll‑independent antibacterial immune response. Nat. Immunol. 2000, 1:342–347. 3‑21 Cytokines made by macrophages and dendritic cells induce a systemic reaction known as the acute‑phase response.


Immunology_Janeway

Immunology_Janeway. 3‑21 Cytokines made by macrophages and dendritic cells induce a systemic reaction known as the acute‑phase response. Bopst, M., Haas, C., Car, B., and Eugster, H.P.: The combined inactivation of tumor necrosis factor and interleukin‑6 prevents induction of the major acute phase proteins by endotoxin. Eur. J. Immunol. 1998, 28:4130–4137. Ceciliani, F., Giordano, A., and Spagnolo, V.: The systemic reaction during inflammation: the acute‑phase proteins. Protein Pept. Lett. 2002, 9:211–223. He, R., Sang, H., and Ye, R.D.: Serum amyloid A induces IL‑8 secretion through a G protein‑coupled receptor, FPRL1/LXA4R. Blood 2003, 101:1572–1581. Horn, F., Henze, C., and Heidrich, K.: Interleukin‑6 signal transduction and lymphocyte function. Immunobiology 2000, 202:151–167. Manfredi, A.A., Rovere-Querini, P., Bottazzi, B., Garlanda, C., and Mantovani, A.: Pentraxins, humoral innate immunity and tissue injury. Curr. Opin. Immunol. 2008, 20:538–544.


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Immunology_Janeway. Manfredi, A.A., Rovere-Querini, P., Bottazzi, B., Garlanda, C., and Mantovani, A.: Pentraxins, humoral innate immunity and tissue injury. Curr. Opin. Immunol. 2008, 20:538–544. Mold, C., Rodriguez, W., Rodic-Polic, B., and Du Clos, T.W.: C‑reactive protein mediates protection from lipopolysaccharide through interactions with FcγR. J. Immunol. 2002, 169:7019–7025. 3‑22 Interferons induced by viral infection make several contributions to host defense. Baldridge, M.T., Nice, T.J., McCune, B.T., Yokoyama, C.C., Kambal, A., Wheadon, M., Diamond, M.S., Ivanova, Y., Artyomov, M., and Virgin, H.W.: Commensal microbes and interferon‑λ determine persistence of enteric murine norovirus infection. Science 2015, 347:266–269. Carrero, J.A., Calderon, B., and Unanue, E.R.: Type I interferon sensitizes lymphocytes to apoptosis and reduces resistance to Listeria infection. J. Exp. Med. 2004, 200:535–540.


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Immunology_Janeway. Carrero, J.A., Calderon, B., and Unanue, E.R.: Type I interferon sensitizes lymphocytes to apoptosis and reduces resistance to Listeria infection. J. Exp. Med. 2004, 200:535–540. Honda, K., Takaoka, A., and Taniguchi, T.: Type I interferon gene induction by the interferon regulatory factor family of transcription factors. Immunity 2006, 25:349–360. Kawai, T., and Akira, S.: Innate immune recognition of viral infection. Nat. Immunol. 2006, 7:131–137. Liu, Y.J.: IPC: professional type 1 interferon‑producing cells and plasmacytoid dendritic cell precursors. Annu. Rev. Immunol. 2005, 23:275–306. Meylan, E., and Tschopp, J.: Toll‑like receptors and RNA helicases: two parallel ways to trigger antiviral responses. Mol. Cell 2006, 22:561–569. Pietras, E.M., Saha, S.K., and Cheng, G.: The interferon response to bacterial and viral infections. J. Endotoxin Res. 2006, 12:246–250.


Immunology_Janeway

Immunology_Janeway. Pietras, E.M., Saha, S.K., and Cheng, G.: The interferon response to bacterial and viral infections. J. Endotoxin Res. 2006, 12:246–250. Pott, J., Mahlakv, T., Mordstein, M., Duerr, C.U., Michiels, T., Stockinger, S., Staeheli, P., and Hornef, M.W.: IFN‑lambda determines the intestinal epithelial antiviral host defense. Proc. Natl Acad. Sci. USA 2011, 108:7944–7949. 3‑23 Several types of innate lymphoid cells provide protection in early infection. Cortez, V.S., Robinette, M.L., and Colonna, M.: Innate lymphoid cells: new insights into function and development. Curr. Opin. Immunol. 2015, 32:71–77. Spits, H., Artis, D., Colonna, M., Diefenbach, A., Di Santo, J.P., Eberl, G., Koyasu, S., Locksley, R.M., McKenzie, A.N., Mebius, R.E., et al.: Innate lymphoid cells—a proposal for uniform nomenclature. Nat. Rev. Immunol. 2013, 13:145–149. 3‑24 NK cells are activated by type I interferon and macrophage‑derived cytokines.


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Immunology_Janeway. 3‑24 NK cells are activated by type I interferon and macrophage‑derived cytokines. Barral, D.C., and Brenner, M.B.: CD1 antigen presentation: how it works. Nat. Rev. Immunol. 2007, 7:929–941. Gineau, L., Cognet, C., Kara, N., Lach, F.P., Dunne, J., Veturi, U., Picard, C., Trouillet, C., Eidenschenk, C., Aoufouchi S., et al.: Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency. J. Clin. Invest. 2012, 122:821–832. Godshall, C.J., Scott, M.J., Burch, P.T., Peyton, J.C., and Cheadle, W.G.: Natural killer cells participate in bacterial clearance during septic peritonitis through interactions with macrophages. Shock 2003, 19:144–149. Lanier, L.L.: Evolutionary struggles between NK cells and viruses. Nat. Rev. Immunol. 2008, 8:259–268. Salazar-Mather, T.P., Hamilton, T.A., and Biron, C.A.: A chemokine‑to‑cytokine‑to‑chemokine cascade critical in antiviral defense. J. Clin. Invest. 2000, 105:985–993.


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Immunology_Janeway. Salazar-Mather, T.P., Hamilton, T.A., and Biron, C.A.: A chemokine‑to‑cytokine‑to‑chemokine cascade critical in antiviral defense. J. Clin. Invest. 2000, 105:985–993. Seki, S., Habu, Y., Kawamura, T., Takeda, K., Dobashi, H., Ohkawa, T., and Hiraide, H.: The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses. Immunol. Rev. 2000, 174:35–46. Yokoyama, W.M., and Plougastel, B.F.: Immune functions encoded by the natural killer gene complex. Nat. Rev. Immunol. 2003, 3:304–316. 3‑25 NK cells express activating and inhibitory receptors to distinguish between healthy and infected cells. & 3‑26 NK‑cell receptors belong to several structural families, the KIRs, KLRs, and NCRs.


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Immunology_Janeway. & 3‑26 NK‑cell receptors belong to several structural families, the KIRs, KLRs, and NCRs. Borrego, F., Kabat, J., Kim, D.K., Lieto, L., Maasho, K., Pena, J., Solana, R., and Coligan, J.E.: Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. Mol. Immunol. 2002, 38:637–660. Boyington, J.C., and Sun, P.D.: A structural perspective on MHC class I recognition by killer cell immunoglobulin‑like receptors. Mol. Immunol. 2002, 38:1007–1021. Brown, M.G., Dokun, A.O., Heusel, J.W., Smith, H.R., Beckman, D.L., Blattenberger, E.A., Dubbelde, C.E., Stone, L.R., Scalzo, A.A., and Yokoyama, W.M.: Vital involvement of a natural killer cell activation receptor in resistance to viral infection. Science 2001, 292:934–937. Long, E.O.: Negative signalling by inhibitory receptors: the NK cell paradigm. Immunol. Rev. 2008, 224:70–84.


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Immunology_Janeway. Long, E.O.: Negative signalling by inhibitory receptors: the NK cell paradigm. Immunol. Rev. 2008, 224:70–84. Robbins, S.H., and Brossay, L.: NK cell receptors: emerging roles in host defense against infectious agents. Microbes Infect. 2002, 4:1523–1530. Trowsdale, J.: Genetic and functional relationships between MHC and NK receptor genes. Immunity 2001, 15:363–374. Vilches, C., and Parham, P.: KIR: diverse, rapidly evolving receptors of innate and adaptive immunity. Annu. Rev. Immunol. 2002, 20:217–251. Vivier, E., Raulet, D.H., Moretta, A., Caligiuri, M.A., Zitvogel, L., Lanier, L.L., Yokoyama, W.M., and Ugolini, S.: Innate or adaptive immunity? The example of natural killer cells. Science 2011, 331:44–49. Vivier, E., Tomasello, E., Baratin, M., Walzer, T., and Ugolini, S.: Functions of natural killer cells. Nat. Immunol. 2008, 9:503–510. 3‑27 NK cells express activating receptors that recognize ligands induced on infected cells or tumor cells.


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Immunology_Janeway. 3‑27 NK cells express activating receptors that recognize ligands induced on infected cells or tumor cells. Brandt, C.S., Baratin, M., Yi, E.C., Kennedy, J., Gao, Z., Fox, B., Haldeman, B., Ostrander, C.D., Kaifu, T., Chabannon, C., et al.: The B7 family member B7‑H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans. J. Exp. Med. 2009, 206:1495–1503. Gasser, S., Orsulic, S., Brown, E.J., and Raulet, D.H.: The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 2005, 436:1186–1190. Gonzalez, S., Groh, V., and Spies, T.: Immunobiology of human NKG2D and its ligands. Curr. Top. Microbiol. Immunol. 2006, 298:121–138. Lanier, L.L.: Up on the tightrope: natural killer cell activation and inhibition. Nat. Immunol. 2008, 9:495–502. Moretta, L., Bottino, C., Pende, D., Castriconi, R., Mingari, M.C., and Moretta, A.: Surface NK receptors and their ligands on tumor cells. Semin. Immunol. 2006, 18:151–158.


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Immunology_Janeway. Moretta, L., Bottino, C., Pende, D., Castriconi, R., Mingari, M.C., and Moretta, A.: Surface NK receptors and their ligands on tumor cells. Semin. Immunol. 2006, 18:151–158. Parham, P.: MHC class I molecules and KIRs in human history, health and survival. Nat. Rev. Immunol. 2005, 5:201–214. Raulet, D.H., and Guerra, N.: Oncogenic stress sensed by the immune system: role of natural killer cell receptors. Nat. Rev. Immunol. 2009, 9:568–580. Upshaw, J.L., and Leibson, P.J.: NKG2D‑mediated activation of cytotoxic lymphocytes: unique signaling pathways and distinct functional outcomes. Semin. Immunol. 2006, 18:167–175. Vivier, E., Nunes, J.A., and Vely, F.: Natural killer cell signaling pathways. Science 2004, 306:1517–1519.


Immunology_Janeway

Immunology_Janeway. Innate immune responses are the body’s initial defense against infection, but these work only to control pathogens that have certain molecular patterns or that induce interferons and other nonspecific defenses. To effectively fight the wide range of pathogens an individual will encounter, the lymphocytes of the adaptive immune system have evolved to recognize a great variety of different antigens from bacteria, viruses, and other disease-causing organisms. An antigen is any molecule or part of a molecule that is specifically recognized by the highly specialized recognition proteins of lymphocytes. On B cells these proteins are the immunoglobulins (Igs), which these cells produce in a vast range of antigen specificities, each B cell producing immunoglobulins of a single specificity (see Section 1-12). A membrane-bound form of immunoglobulin on the B-cell surface serves as the cell’s receptor for antigen, and is known as the B-cell receptor (BCR). A secreted form of immunoglobulin of


Immunology_Janeway

Immunology_Janeway. Section 1-12). A membrane-bound form of immunoglobulin on the B-cell surface serves as the cell’s receptor for antigen, and is known as the B-cell receptor (BCR). A secreted form of immunoglobulin of the same antigen specificity is the antibody produced by terminally differentiated B cells—plasmablasts and plasma cells. The secretion of antibodies, which bind pathogens or their toxic products in the extracellular spaces of the body (see Fig. 1.25), is the main effector function of B cells in adaptive immunity.


Immunology_Janeway

Immunology_Janeway. Antibodies were the first proteins involved in specific immune recognition to be characterized, and are understood in great detail. The antibody molecule has two separate functions: one is to bind specifically to the pathogen or its products that have elicited the immune response; the other is to recruit other cells and molecules to destroy the pathogen once antibody has bound. For example, binding by antibodies can neutralize viruses and mark pathogens for destruction by phagocytes and complement, as described in Chapters 2 and 3. Recognition and effector functions are structurally separated in the antibody molecule, one part of which specifically binds to the antigen whereas the other engages the elimination mechanisms. The antigen-binding region varies extensively between antibody molecules and is known as the variable region or V region. The variability of antibody molecules allows each antibody to bind a different specific antigen, and the total repertoire of antibodies made by a


Immunology_Janeway

Immunology_Janeway. and is known as the variable region or V region. The variability of antibody molecules allows each antibody to bind a different specific antigen, and the total repertoire of antibodies made by a single individual is large enough to ensure that virtually any structure can be recognized. The region of the antibody molecule that engages the effector functions of the immune system does not vary in the same way and


Immunology_Janeway

Immunology_Janeway. The structure of a typical antibody molecule. The interaction of the antibody molecule with specific antigen. Antigen recognition by T cells. is known as the constant region or C region. It comes in five main forms, called isotypes, each of which is specialized for activating different effector mechanisms. The membrane-bound B-cell receptor does not have these effector functions, because the C region remains inserted in the membrane of the B cell. The function of the B-cell receptor is to recognize and bind antigen via the V regions exposed on the surface of the cell, thus transmitting a signal that activates the B cell, leading to clonal expansion and antibody production. To this end, the B-cell receptor is associated with a set of intracellular signaling proteins, which will be described in Chapter 7. Antibodies have become an important class of drug due to their highly specific activities, and we return to discuss their therapeutic uses in Chapter 16.


Immunology_Janeway

Immunology_Janeway. The antigen-recognition molecules of T cells are made solely as membrane-bound proteins, which are associated with an intracellular signaling complex and function only to signal T cells for activation. These T-cell receptors (TCRs) are related to immunoglobulins both in their protein structure—having both V and C regions—and in the genetic mechanism that produces their great variability, which is discussed in Chapter 5. The T-cell receptor differs from the B-cell receptor in an important way, however: it does not recognize and bind antigen by itself, but instead recognizes short peptide fragments of protein antigens that are presented to them by proteins known as MHC molecules on the surface of host cells.


Immunology_Janeway

Immunology_Janeway. The MHC molecules are transmembrane glycoproteins encoded in the large cluster of genes known as the major histocompatibility complex (MHC). The most striking structural feature of MHC molecules is a cleft in the extracellular face of the molecule in which peptides can be bound. MHC molecules are highly polymorphic—each type of MHC molecule occurs in many different versions—within the population. These are encoded by slightly different versions of individual genes called alleles. Most people are therefore heterozygous for the MHC molecules: that is, they express two different alleles for each type of MHC molecule, thus increasing the range of pathogen-derived peptides and self-peptides that can be bound. T-cell receptors recognize features of both the peptide antigen and the MHC molecule to which it is bound. This introduces an extra dimension to antigen recognition by T cells, known as MHC restriction because any given T-cell receptor is specific for a particular peptide bound to a


Immunology_Janeway

Immunology_Janeway. to which it is bound. This introduces an extra dimension to antigen recognition by T cells, known as MHC restriction because any given T-cell receptor is specific for a particular peptide bound to a particular MHC molecule.


Immunology_Janeway

Immunology_Janeway. In this chapter we focus on the structure and antigen-binding properties of immunoglobulins and T-cell receptors. Although B cells and T cells recognize foreign molecules in separate distinct fashions, the receptor molecules they use for this task are very similar in structure. We will see how this basic structure can accommodate great variability in antigen specificity, and how it enables immunoglobulins and T-cell receptors to perform their functions as the anti-gen-recognition molecules of the adaptive immune response. With this foundation, we will return to discuss the impact of MHC polymorphism on T-cell antigen recognition and T-cell development in Chapters 6 and 8, respectively. The structure of a typical antibody molecule.


Immunology_Janeway

Immunology_Janeway. The structure of a typical antibody molecule. Antibodies are the secreted form of the B-cell receptor. Because they are soluble and secreted into the blood in large quantities, antibodies are easily obtained and easily studied. For this reason, most of what we know about the B-cell receptor comes from the study of antibodies. Antibody molecules are roughly Y-shaped, as represented in Fig. 4.1 using three different schematic styles. This part of the chapter will explain how this structure is formed and allows the antibody molecule to perform its dual tasks of binding to a wide variety of antigens while also binding to effector molecules


Immunology_Janeway

Immunology_Janeway. Fig. 4.1 Structure of an antibody molecule. in panel a, the X-ray crystallographic structure of an igg antibody is illustrated as a ribbon diagram of the backbones of the polypeptide chains. The two heavy chains are shown in yellow and purple. The two light chains are both shown in red. Three globular regions form an approximate Y shape. The two antigen-binding sites are at the tips of the arms, which are tethered at their other end to the trunk of the Y by a flexible hinge region. The light-chain variable (VL) and constant region (cL) are indicated. The heavy-chain variable region (Vh) and VL together form the antigen-binding site of the antibody. in panel b, a schematic representation of the same structure denotes each immunoglobulin domain as a separate rectangle. The hinge that tethers each heavy chain’s first constant domain (ch1) to its second (ch2) is illustrated by a thin purple or yellow line, respectively. The antibody-binding sites are indicated by concave regions in VL and


Immunology_Janeway

Immunology_Janeway. each heavy chain’s first constant domain (ch1) to its second (ch2) is illustrated by a thin purple or yellow line, respectively. The antibody-binding sites are indicated by concave regions in VL and Vh. Positions of carbohydrate modifications and disulfide linkages are indicated. in panel c, a more simplified schematic is shown that will be used throughout this book with the variable region in red and the constant region in blue. c terminus, carboxy terminus; n terminus, amino terminus. Structure courtesy of R.L. Stanfield and i.A. Wilson.


Immunology_Janeway

Immunology_Janeway. and to cells that destroy the antigen. Each of these tasks is performed by different parts of the molecule. The ends of the two arms of the Y—the V regions—are involved in antigen binding, and they vary in their detailed structure between different antibody molecules. The stem of the Y—the C region—is far less variable and is the part that interacts with effector molecules and cells. There are five different classes of immunoglobulins, distinguished in their being constructed from C regions that have different structures and properties. These are known as immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE).


Immunology_Janeway

Immunology_Janeway. All antibodies are constructed in the same way from paired heavy and light polypeptide chains, and the generic term immunoglobulin is used for all such proteins. More subtle differences confined to the V region account for the specificity of antigen binding. We will use the IgG antibody molecule as an example to describe the general structural features of immunoglobulins. 4-1 IgG antibodies consist of four polypeptide chains.


Immunology_Janeway

Immunology_Janeway. 4-1 IgG antibodies consist of four polypeptide chains. IgG antibodies are large molecules with a molecular weight of approximately 150 kDa and are composed of two different kinds of polypeptide chains. One, of approximately 50 kDa, is called the heavy or H chain, and the other, of 25 kDa, is the light or L chain (Fig. 4.2). Each IgG molecule consists of two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. In any given immunoglobulin molecule, the two heavy chains and the two light chains are identical, giving an antibody molecule two identical antigen-binding sites. This gives the antibody the ability to bind simultaneously to two identical antigens on a surface, thereby increasing the total strength of the interaction, which is called its avidity. The strength of the interaction between a single antigen-binding site and its antigen is called its affinity.


Immunology_Janeway

Immunology_Janeway. Two types of light chains, lambda (λ) and kappa (κ), are found in antibodies. A given immunoglobulin has either κ chains or λ chains, never one of each. No functional difference has been found between antibodies having λ or κ light chains, and either type of light chain can be found in antibodies of any of the five major classes. The ratio of the two types of light chains varies from species light to species. In mice, the average κ to λ ratio is 20:1, whereas in humans it is 2:1 and in cattle it is 1:20. The reason for this variation is unknown. Distortions of this ratio can sometimes be used to detect the abnormal proliferation of a Fig. 4.2 Immunoglobulin molecules are composed of two types of protein chains: heavy chains and light chains. each immunoglobulin molecule is made up of two hinged heavy chains (green) and two light chains (yellow) joined by disulfide bonds so that each heavy chain is linked to a light chain and the two heavy chains are linked together.


Immunology_Janeway

Immunology_Janeway. B-cell clone, since all progeny of a particular B cell will express an identical light chain. For example, an abnormally high level of λ light chains in a person might indicate the presence of a B-cell tumor that is producing λ chains.


Immunology_Janeway

Immunology_Janeway. The class, and thus the effector function, of an antibody is defined by the structure of its heavy chain. There are five main heavy-chain classes, or isotypes, some of which have several subtypes, and these determine the functional activity of an antibody molecule. The five major classes of immunoglobulin are IgM, IgD, IgG, IgA, and IgE, and their heavy chains are denoted by the lowercase Greek letters μ, δ, γ, α, and ε, respectively. For example, the constant region of IgM is denoted by Cμ. IgG is by far the most abundant immunoglobulin in serum and has several subclasses (IgG1, 2, 3, and 4 in humans). The distinctive functional properties of the different classes and subclasses of antibodies are conferred by the carboxy-terminal part of the heavy chain, where this chain is not associated with the light chain. The general structural features of all the isotypes are similar, particularly with respect to antigen binding. Here we will consider IgG as a typical antibody molecule, and we


Immunology_Janeway

Immunology_Janeway. with the light chain. The general structural features of all the isotypes are similar, particularly with respect to antigen binding. Here we will consider IgG as a typical antibody molecule, and we will return to discuss the structural and functional properties of the different heavy-chain isotypes in Chapter 5.


Immunology_Janeway

Immunology_Janeway. The structure of a B-cell receptor is identical to that of its corresponding antibody except for a small portion of the carboxy terminus of the heavy-chain C region. In the B-cell receptor, the carboxy terminus is a hydrophobic amino acid sequence that anchors the molecule in the membrane, and in the antibody it is a hydrophilic sequence that allows secretion. 4-2 Immunoglobulin heavy and light chains are composed of constant and variable regions.


Immunology_Janeway

Immunology_Janeway. 4-2 Immunoglobulin heavy and light chains are composed of constant and variable regions. The amino acid sequences of many immunoglobulin heavy and light chains have been determined and reveal two important features of antibody molecules. First, each chain consists of a series of similar, although not identical, sequences, each about 110 amino acids long. Each of these repeats corresponds to a discrete, compactly folded region of protein known as an immunoglobulin domain, or Ig domain. The light chain consists of two Ig domains, whereas the heavy chain of the IgG antibody contains four Ig domains (see Fig. 4.2). This suggests that the immunoglobulin chains have evolved by repeated duplication of ancestral gene segments corresponding to a single Ig domain.


Immunology_Janeway

Immunology_Janeway. The second important feature is that the amino-terminal amino acid sequences of the heavy and light chains vary greatly between different antibodies. The variability is limited to approximately the first 110 amino acids, corresponding to the first Ig domain, whereas the remaining domains are constant between immunoglobulin chains of the same isotype. The amino-terminal variable Ig domains (V domains) of the heavy and light chains (VH and VL, respectively) together make up the V region of the antibody and determine its antigen-binding specificity, whereas the constant Ig domains (C domains) of the heavy and light chains (CH and CL, respectively) make up the C region (see Fig. 4.1). The multiple heavy-chain C domains are numbered from the amino-terminal end to the carboxy terminus, for example, CH1, CH2, and so on. 4-3 The domains of an immunoglobulin molecule have similar structures.


Immunology_Janeway

Immunology_Janeway. 4-3 The domains of an immunoglobulin molecule have similar structures. Immunoglobulin heavy and light chains are composed of a series of Ig domains that have a similar overall structure. Within this basic structure, there are distinct differences between V and C domains that are illustrated for the light chain in Fig. 4.3. Each V or C domain is constructed from two β sheets. A β sheet is


Immunology_Janeway

Immunology_Janeway. Fig. 4.3 The structure of immunoglobulin constant and variable domains. The upper panels show schematically the folding pattern of the constant (c) and variable (V) domains of an immunoglobulin light chain. each domain is a barrel-shaped structure in which strands of polypeptide chain (β strands) running in opposite directions (antiparallel) pack together to form two β sheets (shown in yellow and green for the c domain and red and blue for the V domain), which are held together by a disulfide bond. The way in which the polypeptide chain folds to give the final structure can be seen more clearly when the sheets are opened out, as shown in the lower panels. The β strands are lettered sequentially with respect to the order of their occurrence in the amino acid sequence of the domains; the order in each β sheet is characteristic of immunoglobulin domains. The β strands cʹ and cʹʹ that are found in the V domains but not in the c domains are indicated by a blue-shaded background. The


Immunology_Janeway

Immunology_Janeway. the order in each β sheet is characteristic of immunoglobulin domains. The β strands cʹ and cʹʹ that are found in the V domains but not in the c domains are indicated by a blue-shaded background. The characteristic four-strand plus three-strand (c-region type domain) or four-strand plus five-strand (V-region type domain) arrangements are typical immunoglobulin superfamily domain building blocks, found in a whole range of other proteins as well as antibodies and T-cell receptors.


Immunology_Janeway

Immunology_Janeway. built from several β strands, which are regions of protein where several consecutive polypeptides have their peptide backbone bonds arranged in an extended, or flat, conformation. β strands in proteins are sometimes shown as ‘ribbons with an arrow’ to indicate the direction of the polypeptide backbone (see Fig. 4.3). β strands can pack together in a side-by-side manner, being stabilized laterally by two or three backbone hydrogen bonds between adjacent strands. This arrangement is called a β sheet. The Ig domain has two β sheets that are folded onto each other, like two pieces of bread, into a structure called a β sandwich, and are covalently linked by a disulfide bond between cysteine residues from each β sheet. This distinctive structure is known as the immunoglobulin fold.


Immunology_Janeway

Immunology_Janeway. The similarities and differences between V and C domains can be seen in the bottom panels of Fig. 4.3. Here, the Ig domains have been opened out to show how their respective polypeptide chains fold to create each of the βsheets and how each polypeptide chain forms flexible loops between adjacent β strands as it turns to change direction. The main difference between the V and C domains is that the V domain is larger and contains extra β strands, called Cʹ and Cʹʹ. In the V domain, the flexible loops formed between some of the β strands contribute to the antigen-binding site of the immunoglobulin molecule.


Immunology_Janeway

Immunology_Janeway. Many of the amino acids that are common to the C and V domains are present in the core of the immunoglobulin fold and are essential for its stability. Other proteins with sequences similar to those of immunoglobulins have been found to have domains with a similar structure, called immunoglobulin-like domains (Ig-like domains). These domains are present in many proteins of the immune system, such as the KIRs expressed by NK cells described in Chapter 3. They are also frequently involved in cell–cell recognition and adhesion, and together with the immunoglobulins and the T-cell receptors, these proteins make up the extensive immunoglobulin superfamily. 4-4 The antibody molecule can readily be cleaved into functionally distinct fragments.


Immunology_Janeway

Immunology_Janeway. 4-4 The antibody molecule can readily be cleaved into functionally distinct fragments. When fully assembled, an antibody molecule comprises three equal-sized globular portions, with its two arms joined to its trunk by a flexible stretch of polypeptide chain known as the hinge region (see Fig. 4.1b). Each arm of this Y-shaped structure is formed by the association of a light chain with the amino-terminal half of a heavy chain; the VH domain is paired with the VL domain, and the CH1 domain is paired with the CL domain. The two antigen-binding sites are formed by the paired VH and VL domains at the ends of the two arms of the Y (see Fig. 4.1b). The trunk of the Y is formed by the pairing of the carboxyterminal halves of the two heavy chains. The CH3 domains pair with each other but the CH2 domains do not interact. Carbohydrate side chains attached to the CH2 domains lie between the two heavy chains.


Immunology_Janeway

Immunology_Janeway. Proteolytic enzymes (proteases) were an important tool in early studies of antibody structure, and it is valuable to review the terminology they generated. Limited digestion with the protease papain cleaves antibody molecules into three fragments (Fig. 4.4). Papain cuts the antibody molecule on the amino-terminal side of the disulfide bonds that link the two heavy chains, releasing the two arms of the antibody molecule as two identical fragments that contain the antigen-binding activity. These are called the Fab fragments, for fragment antigen binding. The other fragment contains no antigen-binding activity, but because it crystallized readily, it was named the Fc fragment (fragment crystallizable). It corresponds to the paired CH2 and CH3 domains. The Fc fragment is the part of the antibody molecule that does not interact with antigen, but rather interacts with effector molecules and cells, and it differs between heavy-chain isotypes. Another protease, pepsin, cuts on the


Immunology_Janeway

Immunology_Janeway. the antibody molecule that does not interact with antigen, but rather interacts with effector molecules and cells, and it differs between heavy-chain isotypes. Another protease, pepsin, cuts on the carboxy-terminal side of the disulfide bonds (see Fig. 4.4). This produces a fragment, the F(abʹ)2 fragment, in which the two antigen-binding arms of the antibody molecule remain linked. Pepsin cuts the remaining part of the heavy chain into several small fragments. The F(abʹ)2 fragment has exactly the same antigen-binding characteristics as the original antibody but is unable to interact with any effector molecule, such as C1q or Fc receptors, and can be used experimentally to separate the antigen-binding functions from the antibody’s other effector functions.


Immunology_Janeway

Immunology_Janeway. Many antibody-related molecules can be constructed using genetic engineering techniques, and many antibodies and antibody-related molecules are being used therapeutically to treat a variety of diseases. We will return to this topic in Chapter 16, where we discuss the various therapeutic uses of antibodies that have been developed over the last two decades. Fig. 4.4 The Y-shaped immunoglobulin molecule can be dissected by partial digestion with proteases. Upper panels: papain cleaves the immunoglobulin molecule into three pieces, two fab fragments and one fc fragment. The fab fragment contains the V regions and binds antigen. The fc fragment is crystallizable and contains c regions. Lower panels: pepsin cleaves immunoglobulin to yield one f(abʹ)2 fragment and many small pieces of the fc fragment, the largest of which is called the pfcʹ fragment. f(abʹ)2 is written with a prime because it contains a few more amino acids than fab, including the cysteines that form the disulfide bonds.


Immunology_Janeway

Immunology_Janeway. Angle between arms is 90oAngle between arms is 60o(Micrograph ×300,000) 4-5 The hinge region of the immunoglobulin molecule allows flexibility in binding to multiple antigens.


Immunology_Janeway

Immunology_Janeway. The hinge region between the Fc and Fab portions of the IgG molecule allows for some degree of independent movement of the two Fab arms. For example, in the antibody molecule shown in Fig. 4.1a, not only are the two hinge regions clearly bent differently, but the angle between the V and C domains in each of the two Fab arms is also different. This range of motion has led to the junction between the V and C domains being referred to as a ‘molecular ball-and-socket joint.’ This flexibility can be revealed by studies of antibodies bound to small antigens known as haptens. These are molecules of various types that are typically about the size of a tyrosine side chain. Although hap-tens are specifically recognized by antibody, they can stimulate the production of anti-hapten antibodies only when linked to a protein (see Appendix I, Section A-1). Two identical hapten molecules joined by a short flexible region can link two or more anti-hapten antibodies, forming dimers, trimers, tetramers,


Immunology_Janeway

Immunology_Janeway. linked to a protein (see Appendix I, Section A-1). Two identical hapten molecules joined by a short flexible region can link two or more anti-hapten antibodies, forming dimers, trimers, tetramers, and so on, which can be seen by electron microscopy (Fig. 4.5). The shapes formed by these complexes show that antibody molecules are flexible at the hinge region. Some flexibility is also found at the junction between the V and C domains, allowing bending and rotation of the V domain relative to the C domain. Flexibility at both the hinge and the V–C junction enables the two arms of an antibody molecule to bind to sites some distance apart, such as the repeating sites on bacterial cell-wall polysaccharides. Flexibility at the hinge also enables antibodies to interact with the antibody-binding proteins that mediate immune effector mechanisms.


Immunology_Janeway

Summary.

Immunology_Janeway. Summary.


Immunology_Janeway

Immunology_Janeway. The IgG antibody molecule is made up of four polypeptide chains, comprising two identical light chains and two identical heavy chains, and can be thought of as forming a flexible Y-shaped structure. Each of the four chains has a variable (V) region at its amino terminus, which contributes to the antigen-binding site, and a constant (C) region. The light chains are bound to the heavy chains by many noncovalent interactions and by disulfide bonds, and the V regions of the heavy and light chains pair in each arm of the Y to generate two identical antigen-binding sites, which lie at the tips of the arms of the Y. The possession of two antigen-binding sites allows antibody molecules to cross-link antigens and to bind them much more stably and with higher avidity. The trunk of the Y, also called the Fc fragment, is composed of the carboxy-terminal domains of the heavy chains, and it is these domains that determine the antibody’s isotype. Joining the arms of the Y to the trunk are the


Immunology_Janeway

Immunology_Janeway. called the Fc fragment, is composed of the carboxy-terminal domains of the heavy chains, and it is these domains that determine the antibody’s isotype. Joining the arms of the Y to the trunk are the flexible hinge regions. The Fc fragment and hinge regions differ in antibodies of different isotypes.


Immunology_Janeway

Immunology_Janeway. Fig. 4.5 Antibody arms are joined by a flexible hinge. An antigen consisting of two hapten molecules (red balls in diagrams) that can cross-link two antigen-binding sites is used to create antigen:antibody complexes, which can be seen in the electron micrograph. Linear, triangular, and square forms are seen, with short projections or spikes. Limited pepsin digestion removes these spikes (not shown in the figure), which therefore correspond to the fc portion of the antibody; the f(abʹ)2 pieces remain cross-linked by antigen. The interpretation of some of the complexes is shown in the diagrams. The angle between the arms of the antibody molecules varies. in the triangular forms, this angle is 60°, whereas it is 90° in the square forms, showing that the connections between the arms are flexible. Photograph courtesy of n.M. green.


Immunology_Janeway

Immunology_Janeway. Different isotypes have different properties and therefore differ in their interactions with effector molecules and cell types. However, the overall organization of the domains is similar in all isotypes. The interaction of the antibody molecule with specific antigen. In this part of the chapter we look at the antigen-binding site of an immunoglobulin molecule in more detail. We discuss the different ways in which antigens can bind to antibody, and address the question of how variation in the sequences of the antibody V domains determines the specificity for antigen. 4-6 Localized regions of hypervariable sequence form the antigen-binding site.


Immunology_Janeway

Immunology_Janeway. The V regions of any given antibody molecule differ from those of every other. Sequence variability is not, however, distributed evenly throughout the V region but is concentrated in certain segments, as is clearly seen in a variability plot (Fig. 4.6), in which the amino acid sequences of many different antibody V regions are compared. Three particularly variable segments can be identified in both the VH and VL domains. They are designated hypervariable regions and are denoted HV1, HV2, and HV3. In the heavy chains they are located at residues 30 to 36, 49 to 65, and 95 to 103, respectively, while in the light chains they are located at residues 28 to 35, 49 to 59, and 92 to 103, respectively. The most variable part of the domain is in the HV3 region. The regions between the hypervariable regions comprise the rest of the V domain; they show less variability and are termed the framework regions. There are four such regions in each V domain, designated FR1, FR2, FR3, and FR4.


Immunology_Janeway

Immunology_Janeway. The framework regions form the β sheets that provide the structural framework of the immunoglobulin domain. The hypervariable sequences correspond to three loops and are positioned near one another in the folded domain at the outer edge of the β sandwich (Fig. 4.7). Thus, not only is diversity concentrated in particular parts of the V domain sequence, but it is also localized to a particular Fig. 4.6 There are discrete regions of hypervariability in V domains.


Immunology_Janeway

Immunology_Janeway. Fig. 4.6 There are discrete regions of hypervariability in V domains. The hypervariability regions of both the heavy and the light chain contribute to antigen binding of an antibody molecule. A variability plot derived from comparison of the amino acid sequences of several dozen heavy-chain and light-chain V domains is shown. At each amino acid position, the degree of variability is the ratio of the number of different amino acids seen in all of the sequences together to the frequency of the most common amino acid. Three hypervariable regions (hV1, hV2, and hV3) are indicated in red. They are flanked by less variable framework regions (fR1, fR2, fR3, and fR4, shown in blue or yellow). Fig. 4.7 The hypervariable regions lie in discrete loops of the folded structure.


Immunology_Janeway

Immunology_Janeway. Fig. 4.7 The hypervariable regions lie in discrete loops of the folded structure. first panel: the hypervariable regions (red) are positioned on the structure of a map of the coding region of the V domain. Second panel: when shown as a flattened ribbon diagram, hypervariable regions are seen to occur in loops (red) that join particular β strands. Third panel: in the folded structure of the V domain, these loops (red) are brought together to form antigen-binding regions. fourth panel: in a complete antibody molecule, the pairing of a heavy chain and a light chain brings together the hypervariable loops from each chain to create a single hypervariable surface, which forms the antigen-binding site at the tip of each arm. Because they are complementary to the antigen surface, the hypervariable regions are also commonly known as the complementarity-determining regions (cDRs). c, carboxy terminus; n, amino terminus.


Immunology_Janeway

Immunology_Janeway. region on the surface of the molecule. When the VH and VL immunoglobulin domains are paired in the antibody molecule, the three hypervariable loops from each domain are brought together, creating a single hypervariable site at the tip of each arm of the molecule. This is the antigen-binding site, or antibody-combining site, which determines the antigen specificity of the antibody. These six hypervariable loops are more commonly termed the complementarity-determining regions, or CDRs, because the surface they form is complementary to that of the antigen they bind. There are three CDRs from each of the heavy and light chains, namely, CDR1, CDR2, and CDR3. In most cases, CDRs from both VH and VL domains contribute to the antigen-binding site; thus it is the combination of the heavy and the light chain that usually determines the final antigen specificity (see Fig. 4.6). However, there are some Fab crystal structures that show antigen interaction with just the heavy chain; for example, in


Immunology_Janeway

Immunology_Janeway. chain that usually determines the final antigen specificity (see Fig. 4.6). However, there are some Fab crystal structures that show antigen interaction with just the heavy chain; for example, in one influenza Fab, antigen interaction involves binding mostly to the VH CDR3, and only minor contacts with other CDRs. Thus, one way in which the immune system is able to generate antibodies of different specificities is by generating different combinations of heavy-chain and light-chain V regions. This is known as combinatorial diversity; we will encounter a second form of combinatorial diversity in Chapter 5 when we consider how the genes encoding the heavy-chain and light-chain V regions are created from smaller segments of DNA during the development of B cells in the bone marrow.


Immunology_Janeway

Immunology_Janeway. 4-7 Antibodies bind antigens via contacts in CDRs that are complementary to the size and shape of the antigen.


Immunology_Janeway

Immunology_Janeway. In early investigations of antigen binding to antibodies, the only available sources of large quantities of a single type of antibody molecule were tumors of antibody-secreting cells. The antigen specificities of these antibodies were unknown, and therefore many compounds had to be screened to identify ligands that could be used to study antigen binding. In general, the substances found to bind to these antibodies were haptens (see Section 4-5) such as phosphocholine or vitamin K1. Structural analysis of complexes of antibodies with their hapten ligands provided the first direct evidence that the hypervariable regions form the antigen-binding site, and demonstrated the structural basis of specificity for the hapten. Subsequently, with the discovery of methods of generating monoclonal antibodies (see Appendix I, Section A-7), it became possible to make large amounts of pure antibody specific for a given antigen. This has provided a more general picture of how antibodies interact with


Immunology_Janeway

Immunology_Janeway. (see Appendix I, Section A-7), it became possible to make large amounts of pure antibody specific for a given antigen. This has provided a more general picture of how antibodies interact with their antigens, confirming and extending the view of antibody–antigen interactions derived from the study of haptens.


Immunology_Janeway

Immunology_Janeway. The surface of the antibody molecule formed by the juxtaposition of the CDRs of the heavy and light chains is the site to which an antigen binds. The amino acid sequences of the CDRs are different in different antibodies, and so too are the shapes and properties of the surfaces created by these CDRs. As a general principle, antibodies bind ligands whose surfaces are complementary to that of the antigen-binding site. A small antigen, such as a hapten or a short


Immunology_Janeway

Immunology_Janeway. The panels in the top row show schematic representations of the different types of binding sites in a fab fragment of an antibody: first panel, pocket; second panel, groove; third panel, extended surface; and fourth panel, protruding surface. Below are examples of each type. Panel a: the top image shows the molecular surface of the interaction of a small hapten with the complementarity-determining regions (cDRs) of a fab fragment as viewed looking into the antigen-binding site. The ferrocene hapten, shown in red, is bound into the antigen-binding pocket (yellow). in the bottom image (and in those of panels b, c, and d), the molecule has been rotated by about 90° to give a side-on view of the binding site. Panel b: in a complex of an antibody with a peptide from the human immunodeficiency virus (hiV), the peptide (red) binds along a groove (yellow) formed between the heavy-chain and light-chain V domains. Panel c: shown is a complex between hen egg-white lysozyme and the fab fragment


Immunology_Janeway

Immunology_Janeway. virus (hiV), the peptide (red) binds along a groove (yellow) formed between the heavy-chain and light-chain V domains. Panel c: shown is a complex between hen egg-white lysozyme and the fab fragment of its corresponding antibody (hyhel5). The surface on the antibody that comes into contact with the lysozyme is colored yellow. All six cDRs of the antigen-binding site are involved in the binding. Panel d: an antibody molecule against the hiV gp120 antigen has an elongated cDR3 loop (arrow) that protrudes into a recess on the side of the antigen. The structure of the complex between this antibody and gp120 has been solved, and in this case only the heavy chain interacts with gp120. Structures courtesy of R.L. Stanfield and i.A. Wilson.


Immunology_Janeway

Immunology_Janeway. Fig. 4.8 Antigens can bind in pockets, or grooves, or on extended surfaces in the binding sites of antibodies. peptide, generally binds in a pocket or groove lying between the heavy-chain and light-chain V domains (Fig. 4.8a and b). Some antigens, such as proteins, can be the same size as, or larger than, the antibody itself. In these cases, the interface between antigen and antibody is often an extended surface that involves all the CDRs and, in some cases, part of the framework region as well (see Fig. 4.8c). This surface need not be concave but can be flat, undulating, or even convex. In some cases, antibody molecules with elongated CDR3 loops can protrude a ‘finger’ into recesses in the surface of the antigen, as shown in Fig. 4.8d, where an antibody binding to the HIV gp120 antigen projects a long loop into its target. 4-8 Antibodies bind to conformational shapes on the surfaces of antigens using a variety of noncovalent forces.


Immunology_Janeway

Immunology_Janeway. The biological function of antibodies is to bind to pathogens and their products, and to facilitate their removal from the body. An antibody generally recognizes only a small region on the surface of a large molecule such as a polysaccharide or protein. The structure recognized by an antibody is called an antigenic determinant or epitope. Some of the most important pathogens have polysaccharide coats, and antibodies that recognize epitopes formed by the sugar subunits of these molecules are essential in providing immune protection against such pathogens. In many cases, however, the antigens that provoke an immune response are proteins. For example, many protective antibodies against viruses recognize viral coat proteins. In all such cases, the structures recognized by the antibody are located on the surface of the protein. Such sites are likely to be composed of amino acids from different parts of the polypeptide chain that have been brought together by protein folding. Antigenic


Immunology_Janeway

Immunology_Janeway. located on the surface of the protein. Such sites are likely to be composed of amino acids from different parts of the polypeptide chain that have been brought together by protein folding. Antigenic determinants of this kind are known as conformational or discontinuous epitopes because the structure recognized is composed of segments of the protein that are discontinuous in the amino acid sequence of the antigen but are brought together in the three-dimensional structure. In contrast, an epitope composed of a single segment of polypeptide chain is termed a continuous or linear epitope. Although most antibodies raised against intact, fully folded proteins recognize discontinuous epitopes, some will bind to peptide fragments of the protein. Conversely, antibodies raised against peptides of a protein or against synthetic peptides corresponding to part of its sequence are occasionally found to bind to the natural folded protein. This makes it possible, in some cases, to use synthetic


Immunology_Janeway

Immunology_Janeway. of a protein or against synthetic peptides corresponding to part of its sequence are occasionally found to bind to the natural folded protein. This makes it possible, in some cases, to use synthetic peptides in vaccines that aim to raise antibodies against a pathogen’s protein.


Immunology_Janeway

Immunology_Janeway. The interaction between an antibody and its antigen can be disrupted by high salt concentrations, by extremes of pH, by detergents, and sometimes by competition with high concentrations of the pure epitope itself. The binding is therefore a reversible noncovalent interaction. The forces, or bonds, involved in these noncovalent interactions are outlined in Fig. 4.9. Electrostatic interactions occur between charged amino acid side chains, as in salt bridges. Most antibody–antigen interactions involve at least one electrostatic interaction. Interactions also occur between electric dipoles, as in hydrogen bonds, or can involve short-range van der Waals forces. High salt concentrations and extremes of pH disrupt antigen–antibody binding by weakening electrostatic interactions and/or hydrogen bonds. This principle is employed in the purification of antigens by using affinity columns of immobilized antibodies (or in the purification of antibody by using antigens in a like manner)


Immunology_Janeway

Immunology_Janeway. hydrogen bonds. This principle is employed in the purification of antigens by using affinity columns of immobilized antibodies (or in the purification of antibody by using antigens in a like manner) (see Appendix I, Section A-3). Hydrophobic interactions occur when two hydrophobic surfaces come together to exclude water. The strength of a hydrophobic interaction is proportional to the surface area that is hidden from water, and for some antigens, hydrophobic interactions probably account for most of the binding energy. In some cases, water molecules are trapped in pockets in the interface between antigen and antibody. These trapped water molecules, especially those between polar amino acid residues, may also contribute to binding and hence to the specificity of the antibody.


Immunology_Janeway

Immunology_Janeway. The contribution of each of these forces to the overall interaction depends on the particular antibody and antigen involved. A striking difference between antibody interactions with protein antigens and most other natural protein– protein interactions is that antibodies often have many aromatic amino acids HHHHHH HH NH OOC3 N H O C ˜–˜–˜+˜–˜–˜+˜+˜–˜˜–˜–˜–˜–˜–˜––˜+˜+H H O H HO HH O Na+H H O Noncovalent forces Origin Electrostatic forces Hydrogen bonds Attraction between opposite charges Hydrogen shared between electronegative atoms (N, O) Van der Waals forces Hydrophobic forces Cation-pi interaction Fluctuations in electron clouds around molecules polarize neighboring atoms oppositely Hydrophobic groups interact unfavorably with water and tend to pack together to exclude water molecules. The attraction also involves van der Waals forces Non-covalent interaction between a cation and an electron cloud of a nearby aromatic group H H


Immunology_Janeway

Immunology_Janeway. Fig. 4.9 The noncovalent forces that hold together the antigen:antibody complex. Partial charges found in electric dipoles are shown as δ+ or δ–. electrostatic forces diminish as the inverse square of the distance separating the charges, whereas van der Waals forces, which are more numerous in most antigen–antibody contacts, fall off as the sixth power of the separation and therefore operate only over very short ranges. covalent bonds never occur between antigens and naturally produced antibodies.


Immunology_Janeway

Immunology_Janeway. VL VH D1.3 Gln121 in their antigen-binding sites. These amino acids participate mainly in van der Waals and hydrophobic interactions, and sometimes in hydrogen bonds and pi-cation interactions. Tyrosine, for example, can take part in both hydrogen bonding and hydrophobic interactions; it is therefore particularly suitable for providing diversity in antigen recognition and is overrepresented in antigen-binding sites. In general, the hydrophobic and van der Waals forces operate over very short ranges and serve to pull together two surfaces that are complementary in shape: hills on one surface must fit into valleys on the other for good binding to occur. In contrast, electrostatic interactions between charged side chains, and hydrogen bonds bridging oxygen and/or nitrogen atoms, accommodate more specific chemical interactions while strengthening the interaction overall. The side chains of aromatic amino acids such as tyrosine can interact noncovalently through their pi-electron system


Immunology_Janeway

Immunology_Janeway. more specific chemical interactions while strengthening the interaction overall. The side chains of aromatic amino acids such as tyrosine can interact noncovalently through their pi-electron system with nearby cations, including nitrogen-containing side chains that may be in a protonated cationic state.


Immunology_Janeway

Immunology_Janeway. 4-9 Antibody interaction with intact antigens is influenced by steric constraints.


Immunology_Janeway

Immunology_Janeway. An example of an antibody–antigen interaction involving a specific amino acid in the antigen can be seen in the complex of hen egg-white lysozyme with the antibody D1.3 (Fig. 4.10). In this structure, strong hydrogen bonds are formed between the antibody and a particular glutamine in the lysozyme molecule that protrudes between the VH and VL domains. Lysozymes from partridge and turkey have another amino acid in place of the glutamine and do not bind to this antibody. In the high-affinity complex of hen egg-white lysozyme with another antibody, HyHel5 (see Fig. 4.8c), two salt bridges between two basic arginines on the surface of the lysozyme interact with two glutamic acids, one each from the VH CDR1 and CDR2 loops. Lysozymes that lack one of the two arginine residues show a 1000-fold decrease in affinity for HyHel5. Overall surface complementarity must have an important role in antigen–antibody interactions, but in most antibodies that have been studied at this level of detail, only


Immunology_Janeway

Immunology_Janeway. in affinity for HyHel5. Overall surface complementarity must have an important role in antigen–antibody interactions, but in most antibodies that have been studied at this level of detail, only a few residues make a major contribution to the binding energy and hence to the final specificity of the antibody. Although many antibodies naturally bind their ligands with high affinity, in the nanomolar range, genetic engineering by site-directed mutagenesis can tailor an antibody to bind even more strongly to its epitope.


Immunology_Janeway

Immunology_Janeway. Even when antibodies have high affinity for antigens on a larger structure, such as an intact viral particle, antibody binding may be prevented by their particular arrangement. For example, the intact West Nile virion is built from an icosahedral scaffold that has 90 homodimers of a membrane-anchored envelope glycoprotein, E, which has three domains, DI, DII, and DIII. The DIII domain has four polypeptide loops that protrude outward from the viral particle. A neutralizing antibody against West Nile virus, E16, recognizes these loops of DIII, as shown in Fig. 4.11. In theory, there should be 180 possible antigen-binding sites for the E16 antibody on the West Nile viral particle. However, a combination of crystallographic and electron micrographic studies show that even with an excess of the E16 Fab fragment, only about 120 of the total 180 DIII domains of E are able to bind E16 Fab fragment (see Fig. 4.11).


Immunology_Janeway

Immunology_Janeway. Fig. 4.10 The complex of lysozyme with the antibody D1.3. Top panel: The interaction of the fab fragment of D1.3 with hen egg-white lysozyme (heL) is shown. heL is depicted in yellow, the heavy chain (Vh) in turquoise, and the light chain (VL) in green. Bottom panel: A glutamine residue (gln121) that protrudes from heL (yellow) extends its side chain (shown in red) between the VL (green) and Vh (turquoise) domains of the antigen-binding site and makes hydrogen bonds with the hydroxyl group (red dots) of the indicated amino acids of both domains. These hydrogen bonds are important to the antigen–antibody binding. courtesy of R. Mariuzza and R.J. Poljak.


Immunology_Janeway

Immunology_Janeway. Fig. 4.11 Steric hindrance occludes the binding of antibody to native antigen in the intact West Nile viral particle. Top panel: the monoclonal antibody e16 recognizes Diii, one of the three structural domains in the West nile virus glycoprotein e. Shown is a crystal structure of the e16 fab bound to the Diii epitope. Bottom left panel: a computer model was used to dock e16 fab to the mature West nile virion. e16 fabs were able to bind 120 of the 180 Diii epitopes. Sixty of the five-fold clustered Diii epitopes are sterically hindered by the binding of fab to four nearby Diii epitopes. An example of an occluded epitope is the blue area indicated by the arrow. Bottom right panel: cryogenic electron microscopic reconstruction of saturating e16 fab bound to West nile virion confirmed the predicted steric hindrance. The vertices of the triangle shown in the figure indicate the icosahedral symmetry axes.


Immunology_Janeway

Immunology_Janeway. This appears to result from steric hindrance, with the presence of one Fab blocking the ability of another Fab to bind to some nearby E protein sites. Presumably, such steric hindrance would become more severe with intact antibody than is evident with the smaller Fab fragment. This study also showed that the Fab bound to the DIII region using only one of its antigen-binding arms, indicating that antibodies may not always bind to antigens with both antigen-binding sites, depending on the orientation of the antigens being recognized. These constraints will impact the ability of antibodies to neutralize their targets. 4-10 Some species generate antibodies with alternative structures.


Immunology_Janeway

Immunology_Janeway. Our focus in this chapter has been on the structure of antibodies produced by humans, which is generally similar in most mammalian species, including mice, an important model system for immunology research. However, some mammals have the ability to produce an alternative form of antibody that is based on the ability of a single VH domain to interact with antigen in the absence of a VL domain (Fig. 4.12). It has been known for some time that the serum of camels contained abundant immunoglobulin-like material composed of heavy-chain dimers that lack associated light chains but retain the capacity to bind antigens. These antibodies are called heavy-chain-only IgGs (hcIgGs). This property is shared by other camelids, including llamas and alpacas. These species have retained the genes for the immunoglobulin light chains, and some IgG-like material in their sera remains associated with light chains, and so it is unclear what led to this particular adaptation during their evolution. In


Immunology_Janeway

Immunology_Janeway. the immunoglobulin light chains, and some IgG-like material in their sera remains associated with light chains, and so it is unclear what led to this particular adaptation during their evolution. In camelids, the ability to produce hcIgGs arises from mutations that allow the alternative splicing of the heavy-chain mRNA, with loss of the CH1 exon and thus the joining of the VH directly to the CH2 domain in the protein. Other mutations stabilize this structure in the absence of VL domains.


Immunology_Janeway

Immunology_Janeway. Cartilaginous fish, in particular the shark, also have an antibody molecule that differs substantially from human or murine antibodies (see Fig. 4.12). Like camelids, the shark also has genes encoding both immunoglobulin heavy and light chains, and does produce immunoglobulins containing both Fig. 4.12 Camelid and shark antibody can consist of heavy chain only. in the camelid heavy-chain-only antibody, a splicing event of the mature heavy chain can delete the exon encoding the ch1 region and thereby create an in-frame hinge region linking the Vh1 to the ch2 region. in the shark, the heavy-chainonly ig molecule retains the ch1 region, suggesting that this form of antibody may predate the evolution of light chains. for both, the repertoire of antigen-binding sites involves extensive variations in long cDR3 regions of the Vh domain relative to other types of antibody.


Immunology_Janeway

Immunology_Janeway. heavy and light chains. But sharks also produce an immunoglobulin new antigen receptor (IgNAR), with heavy-chain-only antibody in which the VH is spliced to the CH1 exon, rather than the CH1 exon being spliced out as in camelids. These differences suggest that hcIgG production by camelids and sharks represents an event of convergent evolution. The ability of camelid VH domains to interact efficiently with antigens is the basis for producing so-called single-chain antibody. The simplification of using only a single domain for antigen recognition has prompted recent interest in single-chain monoclonal antibodies as an alternative to standard monoclonal antibodies, which we will discuss more in Chapter 16. Summary.


Immunology_Janeway

Immunology_Janeway. X-ray crystallographic analyses of antigen:antibody complexes have shown that the hypervariable loops (complementarity-determining regions, CDRs) of immunoglobulin V regions determine the binding specificity of an antibody. Contact between an antibody molecule and a protein antigen usually occurs over a broad area of the antibody surface that is complementary to the surface recognized on the antigen. Electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic and pi-cation interactions can all contribute to binding. Depending on the size of the antigen, amino acid side chains in most or all of the CDRs make contact with antigen and determine both the specificity and the affinity of the interaction. Other parts of the V region normally play little part in the direct contact with the antigen, but they provide a stable structural framework for the CDRs and help to determine their position and conformation. Antibodies raised against intact proteins usually bind to


Immunology_Janeway

Immunology_Janeway. contact with the antigen, but they provide a stable structural framework for the CDRs and help to determine their position and conformation. Antibodies raised against intact proteins usually bind to the surface of the protein and make contact with residues that are discontinuous in the primary structure of the molecule; they may, however, occasionally bind peptide fragments of the protein, and antibodies raised against peptides derived from a protein can sometimes be used to detect the native protein molecule. Peptides binding to antibodies usually bind in a cleft or pocket between the V regions of the heavy and light chains, where they make specific contact with some, but not necessarily all, of the CDRs. This is also the usual mode of binding for carbohydrate antigens and small molecules such as haptens.


Immunology_Janeway

Immunology_Janeway. Antigen recognition by T cells. In contrast to the immunoglobulins, which interact with pathogens and their toxic products in the extracellular spaces of the body, T cells recognize foreign antigens only when they are displayed on the surface of the body’s own cells. These antigens can derive from pathogens such as viruses or intracellular bacteria, which replicate within cells, or from pathogens or their products that have been internalized by endocytosis from the extracellular fluid.


Immunology_Janeway

Immunology_Janeway. T cells detect the presence of an intracellular pathogen because the infected cells display peptide fragments of the pathogen’s proteins on their surface. These foreign peptides are delivered to the cell surface by specialized host-cell glycoproteins—the MHC molecules. These are encoded in a large cluster of genes that were first identified by their powerful effects on the immune response to transplanted tissues. For that reason, the gene complex was called the major histocompatibility complex (MHC), and the peptide-binding glycoproteins are known as MHC molecules. The recognition of antigen as a small peptide fragment bound to an MHC molecule and displayed at the cell surface is one of the most distinctive features of T cells, and will be the focus of this part of the chapter. How the peptide fragments of antigen are generated and become associated with MHC molecules will be described in Chapter 6.


Immunology_Janeway

Immunology_Janeway. We describe here the structure and properties of the T-cell receptor (TCR). As might be expected from the T-cell receptors’ function as highly variable antigen-recognition structures, the genes for TCRs are closely related to those for immunoglobulins. There are, however, important differences between T-cell receptors and immunoglobulins, and these differences reflect the special features of antigen recognition by T cells. 4-11 The TCRα:β heterodimer is very similar to a Fab fragment of immunoglobulin.


Immunology_Janeway

Immunology_Janeway. T-cell receptors were first identified by using monoclonal antibodies that bound to a single cloned T-cell line: such antibodies either specifically inhibit antigen recognition by the clone or specifically activate it by mimicking the antigen (see Appendix I, Section A-20). These clonotypic antibodies were then used to show that each T cell bears about 30,000 identical antigen receptors on its surface, each receptor consisting of two different polypeptide chains, termed the T-cell receptor α (TCRα) and β (TCRβ) chains. Each chain of the α:β heterodimer is composed of two Ig domains, and the two chains are linked by a disulfide bond, similar to the structure of the Fab fragment of an immunoglobulin molecule (Fig. 4.13). α:β heterodimers account for antigen recognition by most T cells. A minority of T cells bear an alternative, but structurally similar, receptor made up of a different pair of polypeptide chains designated γ and δ. The γ:δ T-cell receptors seem to have different


Immunology_Janeway

Immunology_Janeway. A minority of T cells bear an alternative, but structurally similar, receptor made up of a different pair of polypeptide chains designated γ and δ. The γ:δ T-cell receptors seem to have different antigen-recognition properties from the α:β T-cell receptors, and the functions of γ:δ T cells in immune responses are still being clarified as the various ligands they recognize are identified (see Section 6-20). In the rest of this chapter and elsewhere in the book we use the term T-cell receptor to mean the α:β receptor, except where specified otherwise. Both types of T-cell receptors differ from the membrane-bound immunoglobulin that serves as the B-cell receptor in two main ways. A T-cell receptor has only one antigen-binding site, whereas a B-cell receptor has two, and T-cell receptors are never secreted, whereas immunoglobulins can be secreted as antibodies.


Immunology_Janeway

Immunology_Janeway. Further insights into the structure and function of the α:β T-cell receptor came from studies of cloned cDNA encoding the receptor chains. The amino acid sequences predicted from the cDNA showed that both chains of the T-cell receptor have an amino-terminal variable (V) region with sequence homology to an immunoglobulin V domain, a constant (C) region with homology to an immunoglobulin C domain, and a short stalk segment containing a cysteine residue that forms the interchain disulfide bond (Fig. 4.14). Each chain spans the lipid bilayer by a hydrophobic transmembrane domain, and ends in a short cytoplasmic tail. These close similarities of T-cell receptor chains to the heavy and light immunoglobulin chains first enabled prediction of the structural resemblance of the T-cell receptor heterodimer to a Fab fragment of immunoglobulin.


Immunology_Janeway

Immunology_Janeway. The three-dimensional structure of the T-cell receptor determined by X-ray crystallography in Fig. 4.15a shows that T-cell receptor chains fold in much the same way as the regions comprising the Fab fragment in Fig. 4.1a. Fig. 4.14 Structure of the T-cell receptor. The T-cell receptor heterodimer is composed of two transmembrane glycoprotein chains, α and β. The extracellular portion of each chain consists of two domains, resembling immunoglobulin V and c domains, respectively. Both chains have carbohydrate side chains attached to each domain. A short stalk segment, analogous to an immunoglobulin hinge region, connects the ig-like domains to the membrane and contains the cysteine residue that forms the interchain disulfide bond. The transmembrane helices of both chains are unusual in containing positively charged (basic) residues within the hydrophobic transmembrane segment. The α chain carries two such residues; the β chain has one. Antigen recognition by T cells. 153


Immunology_Janeway

Immunology_Janeway. Antigen recognition by T cells. 153 Fig. 4.13 The T-cell receptor resembles a membrane-bound Fab fragment. The fab fragment of an antibody molecule is a disulfide-linked heterodimer, each chain of which contains one immunoglobulin c domain and one V domain; the juxtaposition of the V domains forms the antigen-binding site (see Section 4-6). The T-cell receptor is also a disulfide-linked heterodimer, with each chain containing an immunoglobulin c-like domain and an immunoglobulin V-like domain. As in the fab fragment, the juxtaposition of the V domains forms the site for antigen recognition.


Immunology_Janeway

Immunology_Janeway. Fig. 4.15 The crystal structure of an α:β T-cell receptor resolved at 0.25 nm. in panels a and b, the α chain is shown in pink and the β chain in blue. Disulfide bonds are shown in green. in panel a, the T-cell receptor is viewed from the side as it would sit on a cell surface, with the cDR loops that form the antigen-binding site (labeled 1, 2, and 3) arrayed across its relatively flat top. in panel b, the cα and cβ domains are shown. The cα domain does not fold into a typical ig-like domain; the face of the domain away from the cβ domain is mainly composed of irregular strands of polypeptide rather than β sheet. The intramolecular disulfide bond (far left) joins a β strand to this segment of α helix. The interaction between the cα and cβ domains is assisted by carbohydrate (colored gray and labeled), with a sugar group from the cα domain making hydrogen bonds to the cβ domain. in panel c, the T-cell receptor is shown aligned with the antigen-binding sites from three different


Immunology_Janeway

Immunology_Janeway. gray and labeled), with a sugar group from the cα domain making hydrogen bonds to the cβ domain. in panel c, the T-cell receptor is shown aligned with the antigen-binding sites from three different antibodies. This view is looking down into the binding site. The Vα domain of the T-cell receptor is aligned with the VL domains of the antigen-binding sites of the antibodies, and the Vβ domain is aligned with the Vh domains. The cDRs of the T-cell receptor and immunoglobulin molecules are colored, with cDRs 1, 2, and 3 of the TcR shown in red and the hV4 loop in orange. for the immunoglobulin V domains, the cDR1 loops of the heavy chain (h1) and light chain (L1) are shown in light and dark blue, respectively, and the cDR2 loops (h2, L2) in light and dark purple, respectively. The heavy-chain cDR3 loops (h3) are in yellow; the light-chain cDR3s (L3) are in bright green. The hV4 loops of the TcR (orange) have no hypervariable counterparts in immunoglobulins. Model structures courtesy of


Immunology_Janeway

Immunology_Janeway. cDR3 loops (h3) are in yellow; the light-chain cDR3s (L3) are in bright green. The hV4 loops of the TcR (orange) have no hypervariable counterparts in immunoglobulins. Model structures courtesy of i.A. Wilson.


Immunology_Janeway

Immunology_Janeway. There are, however, some distinct structural differences between T-cell receptors and Fab fragments. The most striking is in the Cα domain, where the fold is unlike that of any other Ig-like domain. The half of the Cα domain that is juxtaposed with the Cβ domain forms a β sheet similar to that found in other Ig-like domains, but the other half of the domain is formed of loosely packed strands and a short segment of α helix (see Fig. 4.15b). In a Cα domain the intramolecular disulfide bond, which in Ig-like domains normally joins two β strands, joins a β strand to this segment of α helix.


Immunology_Janeway

Immunology_Janeway. There are also differences in the way in which the domains interact. The interface between the V and C domains of both T-cell receptor chains is more extensive than in most antibodies. The interaction between the Cα and Cβ domains is distinctive, as it might be assisted by carbohydrates, with a sugar group from the Cα domain making a number of hydrogen bonds to the Cβ domain (see Fig. 4.15b). Finally, a comparison of the variable binding sites shows that, although the CDR loops align fairly closely with those of antibody molecules, there is some relative displacement (see Fig. 4.15c). This is particularly marked in the Vα CDR2 loop, which is oriented at roughly right angles to the equivalent loop in antibody V domains, as a result of a shift in the β strand that anchors one end of the loop from one face of the domain to the other. A strand displacement also causes a change in the orientation of the Vβ CDR2 loop in some Vβ domains. These differences with antibodies influence the


Immunology_Janeway

Immunology_Janeway. loop from one face of the domain to the other. A strand displacement also causes a change in the orientation of the Vβ CDR2 loop in some Vβ domains. These differences with antibodies influence the ability of the T-cell receptor to recognize their specific ligands, as we will discuss in the next section. In addition to the three hypervariable regions shared with immunoglobulins, the T-cell receptor has a fourth hypervariability region, HV4, in both of its chains (see Fig. 4.15c). These regions occur away from the antigen-binding face of the receptor, and have been implicated in other functions of the TCR, such as superantigen binding, which we will describe in Section 6-14.


Immunology_Janeway

Immunology_Janeway. 4-12 A T-cell receptor recognizes antigen in the form of a complex of a foreign peptide bound to an MHC molecule. Antigen recognition by T-cell receptors clearly differs from recognition by B-cell receptors and antibodies. The immunoglobulin on B cells binds directly to the intact antigen, and, as discussed in Section 4-8, antibodies typically bind to the surface of protein antigens, contacting amino acids that are discontinuous in the primary structure but are brought together in the folded protein. In contrast, αβ T cells respond to short, continuous amino acid sequences. As we described in Section 1-10, these peptide sequences are often buried within the native structure of the protein. Thus, antigens cannot be recognized directly by T-cell receptors unless the protein is unfolded and processed into peptide fragments (Fig. 4.16), and then presented by an MHC molecule (see Fig. 1.15). We will return to the issue of how this process occurs in Chapter 6.


Immunology_Janeway

Immunology_Janeway. The nature of the antigen recognized by T cells became clear with the realization that the peptides that stimulate T cells are recognized only when bound to an MHC molecule. The ligand recognized by the T cell is thus a complex of peptide and MHC molecule. The evidence for involvement of the MHC in T-cell recognition of antigen was at first indirect, but it has been proven conclusively by stimulating T cells with purified peptide:MHC complexes. The T-cell receptor interacts with this ligand by making contacts with both the MHC molecule and the antigen peptide. 4-13 There are two classes of MHC molecules with distinct subunit compositions but similar three-dimensional structures.


Immunology_Janeway

Immunology_Janeway. 4-13 There are two classes of MHC molecules with distinct subunit compositions but similar three-dimensional structures. There are two classes of MHC molecules—MHC class I and MHC class II—and they differ in both their structure and their expression pattern in the tissues of the body. As shown in Figs. 4.17 and 4.18, MHC class I and MHC class II molecules are closely related in overall structure but differ in their subunit composition. In both classes, the two paired protein domains nearest to the membrane resemble immunoglobulin domains, whereas the two domains furthest away from the membrane fold together to create a long cleft, or groove, which is the site at which a peptide binds. Purified peptide:MHC class I and peptide:MHC class II complexes have been characterized structurally, allowing us to describe in detail both the MHC molecules themselves and the way in which they bind peptides.


Immunology_Janeway

Immunology_Janeway. MHC class I molecules (see Fig. 4.17) consist of two polypeptide chains. One chain, the α chain, is encoded in the MHC (on chromosome 6 in humans) and is noncovalently associated with a smaller chain, β2-microglobulin, which is encoded on a different chromosome—chromosome 15 in humans. Only the class I α chain spans the membrane. The complete MHC class I molecule has four domains, three formed from the MHC-encoded α chain, and one contributed by β2-microglobulin. The α3 domain and β2-microglobulin closely resemble Ig-like domains in their folded structure. The folded α1 and α2


Immunology_Janeway

Immunology_Janeway. Fig. 4.16 Differences in the recognition of hen egg-white lysozyme by immunoglobulins and T-cell receptors. Antibodies can be shown by X-ray crystallography to bind epitopes on the surface of proteins, as shown in panel a, where the epitopes for three antibodies are shown in different colors on the surface of hen egg-white lysozyme (see also fig. 4.10). in contrast, the epitopes recognized by T-cell receptors need not lie on the surface of the molecule, because the T-cell receptor recognizes not the antigenic protein itself but a peptide fragment of the protein. The peptides corresponding to two T-cell epitopes of lysozyme are shown in panel b. one epitope, shown in blue, lies on the surface of the protein, but a second, shown in red, lies mostly within the core and is inaccessible in the folded protein. This implies that T-cell receptors do not recognize their epitopes in the context of the native protein. Panel a courtesy of S. Sheriff.


Immunology_Janeway

Immunology_Janeway. domains form the walls of a cleft on the surface of the molecule; because this is where the peptide binds, this part of the MHC molecule is known as the peptide-binding cleft or peptide-binding groove. The MHC molecules are highly polymorphic, and the major differences between the different allelic


Immunology_Janeway

Immunology_Janeway. Fig. 4.17 The structure of an MHC class I molecule determined by X-ray crystallography. Panel a shows a computer graphic representation of a human Mhc class i molecule, hLA-A2, which has been cleaved from the cell surface by the enzyme papain. The surface of the molecule is shown, colored according to the domains shown in panels b–d and described below. Panels b and c show a ribbon diagram of that structure. Shown schematically in panel d, the Mhc class i molecule is a heterodimer of a membrane-spanning α chain (molecular weight 43 kDa) bound noncovalently to β2-microglobulin (12 kDa), which does not span the membrane. The α chain folds into three domains: α1, α2, and α3. The α3 domain and β2-microglobulin show similarities in amino acid sequence to immunoglobulin c domains and have similar folded structures, whereas the α1 and α2 domains are part of the same polypeptide and fold together into a single structure consisting of two separated α helices lying on a sheet of eight


Immunology_Janeway

Immunology_Janeway. similar folded structures, whereas the α1 and α2 domains are part of the same polypeptide and fold together into a single structure consisting of two separated α helices lying on a sheet of eight antiparallel β strands. The folding of the α1 and α2 domains creates a long cleft or groove, which is the site at which peptide antigens bind to the Mhc molecules. for class i molecules, this groove is open at only one end. The transmembrane region and the short stretch of peptide that connects the external domains to the cell surface are not seen in panels a and b because they have been removed by the digestion with papain. As can be seen in panel c, looking down on the molecule from above, the sides of the cleft are formed from the inner faces of the two α helices; the β pleated sheet formed by the pairing of the α1 and α2 domains creates the floor of the cleft.


Immunology_Janeway

Immunology_Janeway. forms are located in the peptide-binding cleft, influencing which peptides will bind and thus the specificity of the dual antigen presented to T cells. By contrast, β2-microglobulin, which does not contribute directly to peptide binding, is not polymorphic.


Immunology_Janeway

Immunology_Janeway. An MHC class II molecule consists of a noncovalent complex of two chains, α and β, both of which span the membrane (see Fig. 4.18). The MHC class II α chain is a different protein from the class I α chain. The MHC class II α and β chains are both encoded within the MHC. The crystallographic structure of the MHC class II molecule shows that it is folded very much like the MHC class I molecule, but the peptide-binding cleft is formed by two domains from different chains, the α1 and β1 domains. The major differences lie at the ends of the peptide-binding cleft, which are more open in MHC class II than in MHC class I molecules. Consequently, the ends of a peptide bound to an MHC class I molecule are substantially buried within the molecule, whereas the ends of peptides bound to MHC class II molecules are not. In both MHC class I and class II molecules, bound peptides are sandwiched between the two α-helical segments of the MHC molecule (Fig. 4.19). The T-cell receptor interacts with this


Immunology_Janeway

Immunology_Janeway. are not. In both MHC class I and class II molecules, bound peptides are sandwiched between the two α-helical segments of the MHC molecule (Fig. 4.19). The T-cell receptor interacts with this compound ligand, making contacts with both the MHC molecule and the peptide antigen. As in the case of MHC class I molecules, the sites of major polymorphism in MHC class II molecules are located in the peptide-binding cleft.


Immunology_Janeway

Immunology_Janeway. Fig. 4.18 MHC class II molecules resemble MHC class I molecules in overall structure. The Mhc class ii molecule is composed of two transmembrane glycoprotein chains, α (34 kDa) and β (29 kDa), as shown schematically in panel d. each chain has two domains, and the two chains together form a compact four-domain structure similar to that of the Mhc class i molecule (compare with panel d of fig. 4.17). Panel a shows a computer graphic representation of the surface of the Mhc class ii molecule, in this case the human protein hLA-DR1, and panel b shows the equivalent ribbon diagram. n, amino terminus; c, carboxy terminus. The α2 and β2 domains, like the α3 and β2-microglobulin domains of the Mhc class i molecule, have amino acid sequence and structural similarities to immunoglobulin c domains; in the Mhc class ii molecule the two domains forming the peptide-binding cleft are contributed by different chains and are therefore not joined by a covalent bond (see panels c and d). Another


Immunology_Janeway

Immunology_Janeway. in the Mhc class ii molecule the two domains forming the peptide-binding cleft are contributed by different chains and are therefore not joined by a covalent bond (see panels c and d). Another important difference, not apparent in this diagram, is that the peptide-binding groove of the Mhc class ii molecule is open at both ends.


Immunology_Janeway

Immunology_Janeway. Fig. 4.19 MHC molecules bind peptides tightly within the cleft. When Mhc molecules are crystallized with a single synthetic peptide antigen, the details of peptide binding are revealed. in Mhc class i molecules (panels a and c), the peptide is bound in an elongated conformation with both ends tightly bound at either end of the cleft. in Mhc class ii molecules (panels b and d), the peptide is also bound in an elongated conformation but the ends of the peptide are not tightly bound and the peptide extends beyond the cleft. The upper surface of the peptide:Mhc complex is recognized by T cells, and is composed of residues of the Mhc molecule and the peptide. The amino acid side chains of the peptide insert into pockets in the peptide-binding groove of the Mhc molecule; these pockets are lined with residues that are polymorphic within the Mhc. in representations c and d, the surfaces of the different pockets for the different amino acids are depicted as areas of different colors.


Immunology_Janeway

Immunology_Janeway. are lined with residues that are polymorphic within the Mhc. in representations c and d, the surfaces of the different pockets for the different amino acids are depicted as areas of different colors. Structures courtesy of


Immunology_Janeway

Immunology_Janeway. R.L. Stanfield and i.A. Wilson. 4-14 Peptides are stably bound to MHC molecules, and also serve to stabilize the MHC molecule on the cell surface. An individual can be infected by a wide variety of pathogens, whose proteins will not generally have peptide sequences in common. For T cells to be able to detect the widest possible array of infections, the MHC molecules (both class I and class II) of an individual should be able to bind stably to many different peptides. This behavior is quite distinct from that of other peptide-binding receptors, such as those for peptide hormones, which usually bind only a single type of peptide. The crystal structures of peptide:MHC complexes have helped to show how a single binding site can bind a peptide with high affinity while retaining the ability to bind a wide variety of different peptides.


Immunology_Janeway

Immunology_Janeway. An important feature of the binding of peptides to MHC molecules is that the peptide is bound as an integral part of the MHC molecule’s structure, and MHC molecules are unstable when peptides are not bound. This dependence on bound peptide applies to both MHC class I and MHC class II molecules. Stable peptide binding is important, because otherwise peptide exchanges occurring at the cell surface would prevent peptide:MHC complexes from being reliable indicators of infection or of uptake of a specific antigen. When MHC molecules are purified from cells, their stably bound peptides co-purify with them, and this fact has enabled the peptides bound by particular MHC molecules to be analyzed. Peptides are released from the MHC molecules by denaturing the complex in acid, and they are then purified and sequenced. Pure synthetic peptides can also be incorporated into empty MHC molecules and the structure of the complex determined, revealing details of the contacts between the MHC molecule


Immunology_Janeway

Immunology_Janeway. and sequenced. Pure synthetic peptides can also be incorporated into empty MHC molecules and the structure of the complex determined, revealing details of the contacts between the MHC molecule and the peptide. From such studies a detailed picture of the binding interactions has been built up. We first discuss the peptide-binding properties of MHC class I molecules.


Immunology_Janeway

Immunology_Janeway. 4-15 MHC class I molecules bind short peptides of 8–10 amino acids by both ends.


Immunology_Janeway

Immunology_Janeway. Binding of a peptide to an MHC class I molecule is stabilized at both ends of the peptide-binding cleft by contacts between atoms in the free amino and carboxy termini of the peptide and invariant sites that are found at each end of the cleft in all MHC class I molecules (Fig. 4.20). These are thought to be the main stabilizing contacts for peptide:MHC class I complexes, because synthetic peptide analogs lacking terminal amino and carboxyl groups fail to bind stably to MHC class I molecules. Other residues in the peptide serve as additional anchors. Peptides that bind to MHC class I molecules are usually 8–10 amino acids long. Longer peptides are thought to bind, however, particularly if they can bind at their carboxy terminus, but they are subsequently shortened to 8–10 amino acids through cleavage by exopeptidases present in the endoplasmic reticulum, which is where MHC class I molecules bind peptides. The peptide lies in an elongated conformation along the cleft; variations in


Immunology_Janeway

Immunology_Janeway. cleavage by exopeptidases present in the endoplasmic reticulum, which is where MHC class I molecules bind peptides. The peptide lies in an elongated conformation along the cleft; variations in peptide length seem to be accommodated, in most cases, by a kinking in the peptide backbone. However, in some cases, length variation can also be accommodated in MHC class I molecules by allowing the peptide to extend out of the cleft at the carboxy terminus.


Immunology_Janeway

Immunology_Janeway. These interactions give MHC class I molecules a broad peptide-binding specificity. In addition, MHC molecules are highly polymorphic. As mentioned earlier, MHC genes are highly polymorphic and there are hundreds of different allelic variations of the MHC class I genes in the human population. Each individual carries only a small selection of these variants. The main differences between allelic MHC variants are found at certain sites in the peptide-binding cleft, resulting in different amino acids in key peptide-interaction sites. Because of this, different MHC variants preferentially bind different peptides. The peptides that can bind to a given MHC variant have the same or very similar amino acid residues at two or three particular positions along the peptide sequence. The amino acid side chains at these positions insert into pockets in the MHC molecule that are lined by the polymorphic amino acids. Because this binding anchors the peptide to the MHC molecule, the peptide residues


Immunology_Janeway

Immunology_Janeway. chains at these positions insert into pockets in the MHC molecule that are lined by the polymorphic amino acids. Because this binding anchors the peptide to the MHC molecule, the peptide residues involved are called the anchor residues, as illustrated in Fig. 4.21. Both the position and identity of these anchor residues can vary, depending on the particular MHC class I variant that is binding the peptide. However, most peptides that bind to MHC class I molecules have a hydrophobic (or sometimes basic) residue at the carboxy terminus that also serves to anchor the peptide in the groove. Whereas changing an anchor residue will in most cases prevent the peptide from binding, not every synthetic peptide of suitable length that


Immunology_Janeway

Immunology_Janeway. Antigen recognition by T cells. 159 Fig. 4.20 Peptides are bound to MHC class I molecules by their ends. Mhc class i molecules interact with the backbone of a bound peptide (shown in yellow) through a series of hydrogen bonds and ionic interactions (shown as dotted blue lines) at each end of the peptide. The amino terminus of the peptide is to the left, the carboxy terminus to the right. Black circles are carbon atoms; red are oxygen; blue are nitrogen. The amino acid residues in the Mhc molecule that form these bonds are common to all Mhc class i molecules, and their side chains are shown in full (in gray) on a ribbon diagram of the Mhc class i groove. A cluster of tyrosine residues common to all Mhc class i molecules forms hydrogen bonds to the amino terminus of the bound peptide, while a second cluster of residues forms hydrogen bonds and ionic interactions with the peptide backbone at the carboxy terminus and with the carboxy terminus itself.


Immunology_Janeway

Immunology_Janeway. Fig. 4.21 Peptides bind to MHC molecules through structurally related anchor residues. Peptides eluted from two different Mhc class i molecules are shown in the upper and lower panels, respectively. The anchor residues (green) differ for peptides that bind different allelic variants of Mhc class i molecules but are similar for all peptides that bind to the same Mhc molecule. The anchor residues that bind a particular Mhc molecule need not be identical, but are always related: for example, phenylalanine (f) and tyrosine (Y) are both aromatic amino acids, whereas valine (V), leucine (L), and isoleucine (i) are all large hydrophobic amino acids. Peptides also bind to Mhc class i molecules through their amino (blue) and carboxy (red) termini.


Immunology_Janeway

Immunology_Janeway. Fig. 4.22 Peptides bind to MHC class II molecules by interactions along the length of the binding groove. A peptide (yellow; shown as the peptide backbone only, with the amino terminus to the left and the carboxy terminus to the right) is bound by an Mhc class ii molecule through a series of hydrogen bonds (dotted blue lines) that are distributed along the length of the peptide. The hydrogen bonds toward the amino terminus of the peptide are made with the backbone of the Mhc class ii polypeptide chain, whereas throughout the peptide’s length bonds are made with residues that are highly conserved in Mhc class ii molecules. The side chains of these residues are shown in gray on the ribbon diagram of the Mhc class ii groove.


Immunology_Janeway

Immunology_Janeway. contains these anchor residues will bind the appropriate MHC class I molecule, and so the overall binding must also depend on the nature of the amino acids at other positions in the peptide. In some cases, particular amino acids are preferred in certain positions, whereas in others the presence of particular amino acids prevents binding. These additional amino acid positions are called ‘secondary anchors.’ These features of peptide binding enable an individual MHC class I molecule to bind a wide variety of different peptides, yet allow different MHC class I allelic variants to bind different sets of peptides. As we will see in Chapter 15, MHC polymorphisms also impact the binding of peptides derived from self-proteins and can influence the susceptibility of an individual to various autoimmune diseases. 4-16 The length of the peptides bound by MHC class II molecules is not constrained.