8536d_ch04_076-104 9/6/029:02 PM Page 81 maces Mac 85: 365_smmboldsby et al./ Immunology Se Antibodies: Structure and Function CHAPTER 4 81 FIGURE 4-5 Ribbon representation of an intact monoclonal anti- gion. [The laboratory of A. McPherson provided this image, which is body depicting the heavy chains (yellow and blue) and light chains based on x- ray crystallography data determined by L. Harris et al red). The domains of the molecule composed of B pleated sheets 1992, Nature 360: 369. The image was generated using the computer are readily visible as is the extended conformation of the hinge re- ram RIBBONS. across the faces of the B sheets(Figure 4-8). Interactions sequences of amino acids that form the loops connecting form links between identical domains (e.g, CH2/CH2, the B strands. As the next section explains, some of the CH3/CH3, and CH4/CH4)and between nonidentical do- loop sequences of the VH and Vi domains contain variable mains(e. g, VH/VL and CHI/CL). The structure of the im- amino acids and constitute the antigen-binding site of the munoglobulin fold also allows for variable lengths and molecule (a)y,6.a No hinge dditional Biological cHo RE4-6 (a)Heavy and light chains are folded into domains, effector functions are mediated by the other domains (b)The u and ntaining about 110 amino acid residues and an intrachain e heavy chains contain an additional domain that replaces the hinge disulfide bond that forms a loop of 60 amino acids. The amino. region terminal domains, corresponding to the V regions, bind to antigen;
across the faces of the sheets (Figure 4-8). Interactions form links between identical domains (e.g., CH2/CH2, CH3/CH3, and CH4/CH4) and between nonidentical domains (e.g., VH/VL and CH1/CL). The structure of the immunoglobulin fold also allows for variable lengths and sequences of amino acids that form the loops connecting the strands. As the next section explains, some of the loop sequences of the VH and VL domains contain variable amino acids and constitute the antigen-binding site of the molecule. Antibodies: Structure and Function CHAPTER 4 81 FIGURE 4-5 Ribbon representation of an intact monoclonal antibody depicting the heavy chains (yellow and blue) and light chains (red). The domains of the molecule composed of pleated sheets are readily visible as is the extended conformation of the hinge reFIGURE 4-6 (a) Heavy and light chains are folded into domains, each containing about 110 amino acid residues and an intrachain disulfide bond that forms a loop of 60 amino acids. The aminoterminal domains, corresponding to the V regions, bind to antigen; gion. [The laboratory of A. McPherson provided this image, which is based on x-ray crystallography data determined by L. J. Harris et al., 1992, Nature 360:369. The image was generated using the computer program RIBBONS.] CHO S S S S S S S S S S S S S S S S S S S S S S S S CH2 (a) γ, δ, α (b) , CH3 CHO Hinge 261 321 367 425 446 214 200 194 144 134 22 CH1 VH CL VL S S S S S S S S Biological activity No hinge region Antigen binding 88 CH2 CH3 CH4 Additional domain effector functions are mediated by the other domains. (b) The and heavy chains contain an additional domain that replaces the hinge region. 8536d_ch04_076-104 9/6/02 9:02 PM Page 81 mac85 Mac 85:365_smm:Goldsby et al. / Immunology 5e:
8536d_ch04_076-104 9/5/02 6: 19 AM Page 82 mac76 mac76: 385 Goldapy et al/Immunologyse: 2 PART II Generation of B-Cell and T-Cell Respons Cdomain B strands COOH Disulfide bond coo OOH CDRs FICURE4-7(a)Diagram of an immunoglobulin light chain depict. CDRs (complementarity-determining regions). Heavy-chain do- munoglobulin-fold structure of its variable and constant mains have the same characteristic structure. (b) The B pleated domains. The two p pleated sheets in each domain are held together sheets are opened out to reveal the relationship of the individual B by hydrophobic interactions and the conserved disulfide bond. The p strands and joining loops. Note that the variable domain contains strands that compose each sheet are shown in different colors. The two more p strands than the constant domain [Part(a) adapted amino acid sequences in three loops of each variable domain show from M. Schiffer et al, 1973, Biochemistry 12: 4620; reprinted with considerable variation; these hypervariable regions(blue) make up permission; part(b ) adapted from Williams and Barclay, 1988, Annu the antigen-binding site. Hypervariable regions are usually called Rev Immunol. 6: 381.1 Diversity in the Variable-Region Domain Thus if a comparison of the sequences of 100 heavy chains Is Concentrated in CDRs revealed that a serine was found in position 7 in 51 of the se quences(frequency 0.51), it would be the most common Detailed comparisons of the amino acid sequences of a large amino acid. If examination of the other 49 sequences showed number of Vu and VH domains revealed that the sequence that position 7 was occupied by either glutamine, histidine, variation is concentrated in a few discrete regions of these proline, or tryptophan, the variability at that position would domains. The pattern of this variation is best summarized by be 9.8(5/0.51) Variability plots of Vi and VH domains of hu a quantitative plot of the variability at each position of the man antibodies show that maximum variation is seen in polypeptide chain. The variability is defined those portions of the sequence that correspond to the loops that join the B strands( Figure 4-9). These regions were orig- of different amino acids at a given position ally called hypervariable regions in recognition of their variab igh variability. Hypervariable regions form the antiger Frequency of the most common amino acid binding site of the antibody molecule. Because the antigen binding site is complementary to the structure of the epitope
Diversity in the Variable-Region Domain Is Concentrated in CDRs Detailed comparisons of the amino acid sequences of a large number of VL and VH domains revealed that the sequence variation is concentrated in a few discrete regions of these domains. The pattern of this variation is best summarized by a quantitative plot of the variability at each position of the polypeptide chain. The variability is defined as: # of different amino acids at a given position Variability Frequency of the most common amino acid at given position Thus if a comparison of the sequences of 100 heavy chains revealed that a serine was found in position 7 in 51 of the sequences (frequency 0.51), it would be the most common amino acid. If examination of the other 49 sequences showed that position 7 was occupied by either glutamine, histidine, proline, or tryptophan, the variability at that position would be 9.8 (5/0.51). Variability plots of VL and VH domains of human antibodies show that maximum variation is seen in those portions of the sequence that correspond to the loops that join the strands (Figure 4-9). These regions were originally called hypervariable regions in recognition of their high variability. Hypervariable regions form the antigenbinding site of the antibody molecule. Because the antigen binding site is complementary to the structure of the epitope, 82 PART II Generation of B-Cell and T-Cell Responses FIGURE 4-7 (a) Diagram of an immunoglobulin light chain depicting the immunoglobulin-fold structure of its variable and constant domains. The two pleated sheets in each domain are held together by hydrophobic interactions and the conserved disulfide bond. The strands that compose each sheet are shown in different colors. The amino acid sequences in three loops of each variable domain show considerable variation; these hypervariable regions (blue) make up the antigen-binding site. Hypervariable regions are usually called (a) (b) CL domain Disulfide bond β strands β-strand arrangement Loops VL domain NH2 NH2 COOH COOH COOH CDRs CDRs NH2 CDRs (complementarity-determining regions). Heavy-chain domains have the same characteristic structure. (b) The pleated sheets are opened out to reveal the relationship of the individual strands and joining loops. Note that the variable domain contains two more strands than the constant domain. [Part (a) adapted from M. Schiffer et al., 1973, Biochemistry 12:4620; reprinted with permission; part (b) adapted from Williams and Barclay, 1988, Annu. Rev. Immunol. 6:381.] 8536d_ch04_076-104 9/5/02 6:19 AM Page 82 mac76 mac76:385 Goldsby et al./Immunology5e:
8536d_ch04076-104 9/6/02 9:02 PM Page 83 macas Mac 85:365 smm polsby et al. / Immunology se Antibodies: Structure and Function chapter 4 VH domain VH domain C domain Antigen-binding site drate chain VI domain Heavy chains Carbohydrate FICURE4-8Interactions between domains in the separate chains teracting heavy- and light-chain domains. Note that the CH2/CH2 of an immunoglobulin molecule are critical to its quaternary struc. domains protrude because of the presence of carbohydrate(tan)in ture.(a)Model of IgG molecule, based on x-ray crystallographic the interior. The protrusion makes this domain more accessible, en- analysis, showing associations between domains. Each solid ball rep bling it to interact with molecules such as certain complement resents an amino acid residue; the larger tan balls are carbohydrate. components. Part(a) from E. W. Silverton et al,1977, Proc.Nat. The two light chains are shown in shades of red; the two heavy Acad. Sci. U.S.A. 74: 5140. 1 chains, in shades of blue. (b) A schematic diagram showing the in- these areas are now more widely called complementarity de- analyzed to date can be superimposed on one another; in termining regions( CDRs). The three heavy-chain and three contrast, the hypervariable loops(i. e, the CDRs)have differ light-chain CDR regions are located on the loops that con- ent orientations in different antibodies nect the b strands of the VH and Vl domains. The remainde of the Vi and vh domains exhibit far less variation; the stretches are called the framework regions(FRs). The wide CDRs Bind Antigen range of specificities exhibited by antibodies is due to varia- The finding that CDRs are the antigen-binding regions of tions in the length and amino acid sequence of the six CDRs antibodies has been confirmed directly by high-resolution in each Fab fragment. The framework region acts as a scaf- x-ray crystallography of antigen-antibody complexes. Crys fold that supports these six loops. The three-dimensional tallographic analysis has been completed for many Fab structure of the framework regions of virtually all antibodies fragments of monoclonal antibodies complexed either with Go to www.whfreeman.com/immunology6molecularVisualization Antibody Recognition of Antigen
these areas are now more widely called complementarity determining regions (CDRs). The three heavy-chain and three light-chain CDR regions are located on the loops that connect the strands of the VH and VL domains. The remainder of the VL and VH domains exhibit far less variation; these stretches are called the framework regions (FRs). The wide range of specificities exhibited by antibodies is due to variations in the length and amino acid sequence of the six CDRs in each Fab fragment. The framework region acts as a scaffold that supports these six loops. The three-dimensional structure of the framework regions of virtually all antibodies analyzed to date can be superimposed on one another; in contrast, the hypervariable loops (i.e., the CDRs) have different orientations in different antibodies. CDRs Bind Antigen The finding that CDRs are the antigen-binding regions of antibodies has been confirmed directly by high-resolution x-ray crystallography of antigen-antibody complexes. Crystallographic analysis has been completed for many Fab fragments of monoclonal antibodies complexed either with Antibodies: Structure and Function CHAPTER 4 83 FIGURE 4-8 Interactions between domains in the separate chains of an immunoglobulin molecule are critical to its quaternary structure. (a) Model of IgG molecule, based on x-ray crystallographic analysis, showing associations between domains. Each solid ball represents an amino acid residue; the larger tan balls are carbohydrate. The two light chains are shown in shades of red; the two heavy chains, in shades of blue. (b) A schematic diagram showing the inVL domain Antigen–binding site CL domain Heavy chains Carbohydrate chain Carbohydrate Antigen–binding site VH domain (a) (b) S S VH CL VL CΗ2 CΗ3 V CΗ1 H CΗ2 VL VL domain VH domain CH1 CH2 CH3 teracting heavy- and light-chain domains. Note that the CH2/CH2 domains protrude because of the presence of carbohydrate (tan) in the interior. The protrusion makes this domain more accessible, enabling it to interact with molecules such as certain complement components. [Part (a) from E. W. Silverton et al., 1977, Proc. Nat. Acad. Sci. U.S.A. 74:5140.] Go to www.whfreeman.com/immunology Molecular Visualization Antibody Recognition of Antigen 8536d_ch04_076-104 9/6/02 9:02 PM Page 83 mac85 Mac 85:365_smm:Goldsby et al. / Immunology 5e:
8536d_ch04_076-1049/6/02 9: 02 PM Page 84 maca Mac 85: 365_smm pldsby et al./ Immunology Se 4 PART I1 Generation of B-Cell and T-Cell Response omain V domain CDRI CDR CDR3 CDRI CDR2 CDR3 100 40 100 Residue position number Residue position number FIGURE4. Variability of amino acid residues in the V, and VH do. light-chain V domain are brought into proximity in the folded struc mains of human antibodies with different specificities. Three hyper- ture. The same is true of the heavy-chain V domain. Based on E.A. variable(HV) regions, also called complementarity-determining Kabat et al., 1977, Sequence of Immunoglobulin Chains, U.SDept mains. As shown in Figure 4-7(right), the three HV regions in the of Health Education, and Welfare. J regions(CDRs), are present in both heavy- and light-chain V do large globular protein antigens or with a number of smaller shown that several CDRs may make contact with the antigen, antigens including carbohydrates, nucleic acids, peptides, and a number of complexes have been observed in which all and small haptens. In addition, complete structures have six CDRs contact the antigen. In general, more residues in the been obtained for several intact monoclonal antibodies. X- heavy-chain CDRs appear to contact antigen than in the ray diffraction analysis of antibody-antigen complexes has light-chain CDRs. Thus the VH domain often contributes o(a)Side view of the three-dimensional structure of Waals contact of the angiotensin peptide. (b) Side view of the van the combining site of an angiotensin -Fab complex. The peptide is der Waals surface of contact between angiotensin ll and Fab frag- in red. The three heavy-chain CDRS(H1, H2, H3)and three light- ment [ From K. C. Garcia et al, 1992, Science 257: 502: courtesy of chain CDRs(L1, L2, L3)are each shown in a different color. All six M. Amzel, Johns Hopkins University I CDRs contain side chains, shown in yellow, that are within van der
large globular protein antigens or with a number of smaller antigens including carbohydrates, nucleic acids, peptides, and small haptens. In addition, complete structures have been obtained for several intact monoclonal antibodies. Xray diffraction analysis of antibody-antigen complexes has shown that several CDRs may make contact with the antigen, and a number of complexes have been observed in which all six CDRs contact the antigen. In general, more residues in the heavy-chain CDRs appear to contact antigen than in the light-chain CDRs. Thus the VH domain often contributes 84 PART II Generation of B-Cell and T-Cell Responses Residue position number VL domain 150 0 80 20 100 40 60 120 Variability 100 50 0 CDR1 CDR2 CDR3 Variability 150 0 25 50 75 100 120 60 30 0 Residue position number VH domain CDR1 CDR2 CDR3 FIGURE 4-9 Variability of amino acid residues in the VL and VH domains of human antibodies with different specificities. Three hypervariable (HV) regions, also called complementarity-determining regions (CDRs), are present in both heavy- and light-chain V domains. As shown in Figure 4-7 (right), the three HV regions in the light-chain V domain are brought into proximity in the folded structure. The same is true of the heavy-chain V domain. [Based on E. A. Kabat et al., 1977, Sequence of Immunoglobulin Chains, U.S. Dept. of Health, Education, and Welfare.] (a) (b) FIGURE 4-10 (a) Side view of the three-dimensional structure of the combining site of an angiotensin II–Fab complex. The peptide is in red. The three heavy-chain CDRs (H1, H2, H3) and three lightchain CDRs (L1, L2, L3) are each shown in a different color. All six CDRs contain side chains, shown in yellow, that are within van der Waals contact of the angiotensin peptide. (b) Side view of the van der Waals surface of contact between angiotensin II and Fab fragment. [From K. C. Garcia et al., 1992, Science 257:502; courtesy of M. Amzel, Johns Hopkins University.] 8536d_ch04_076-104 9/6/02 9:02 PM Page 84 mac85 Mac 85:365_smm:Goldsby et al. / Immunology 5e:
8536a_ch04-076-104 9/5/02 6: 19 AM Page 85 mac76 mac76: 385 Goldaby et al/Immunologyse: Antibodies Structure and Function CHAPTER 4 85 more to antigen binding than the Vi domain. The dominant small octapeptide hormone angiotensin II with the binding role of the heavy chain in antigen binding was demonstrated site of an anti-angiotensin antibody( Figure 4-10) in a study in which a single heavy chain specific for a glyco protein antigen of the human immunodeficiency virus Conformational Changes May Be antigenic specificity. All of the hybrid antibodies bound to Induced by Antigen Binding the hiv glycoprotein antigen, indicating that the heavy chain As more x-ray crystallographic analyses of Fab fragments alone was sufficient to confer specificity. However, one were completed, it became clear that in some cases binding of should not conclude that the light chain is largely irrelevant; antigen induces conformational changes in the antibody in some antibody-antigen reactions, the light chain makes antigen, or both. Formation of the complex between neur- the more important contribution. aminidase and anti-neuraminidase is accompanied by a The actual shape of the antigen binding site formed by change in the orientation of side chains of both the epitope whatever combination of CDRs are used in a particular anti- and the antigen-binding site. This conformational change re body has been shown to vary dramatically. As pointed out in sults in a closer fit between the epitope and the antibodys Chapter 3, contacts between a large globular protein antigen binding site and antibody occur over a broad, often rather flat, undulat- In another example, comparison of an anti-hemagglutin ing face. In the area of contact, protrusions or depressions on Fab fragment before and after binding to a hemagglutinin the antigen are likely to match complementary depressions peptide antigen has revealed a visible conformational chang or protrusions on the antibody In the case of the well studied in the heavy-chain CDR3 loop and in the accessible surface of lysozyme/anti-lysozyme system, crystallographic studies the binding site. Another striking example of conformational have shown that the surface areas of interaction are quite change has been seen in the complex between an Fab frag- large, ranging from about 650 A2 to more than 900 A ment derived from a monoclonal antibody against the Hiv Within this area, some 15-22 amino acids in the antibody protease and the peptide epitope of the protease As shown in contact the same number of residues in the protein antigen. Figure 4-11, there are significant changes in the Fab upon In contrast, antibodies bind smaller antigens, such as small binding. In fact, upon antigen binding, the CDRI region of haptens, in smaller, recessed pockets in which the ligand is the light chain moves as much as I A and the heavy chain buried. This is nicely illustrated by the interaction of the CDR3 moves 2.7 A. Thus, in addition to variability in the FIGURE4-11 Structure of a complex between a peptide derived line shows its structure when bound. There are significant confo from HIV protease and an Fab fragment from an anti-protease anti- mational changes in the DRs of the Fab on binding the antigen ody (left)and comparison of the Fab structure before and after pep. These are especially pronounced in the light chain CDRI(L1)and tide binding (right). In the right panel, the red line shows the the heavy chain CDR3(H3) From]. Lescar et al., 1997, ) Mol Biol structure of the Fab fragment before it binds the peptide and the blue 267: 1207: courtesy of G. Bentley, Institute Pasteur I
more to antigen binding than the VL domain. The dominant role of the heavy chain in antigen binding was demonstrated in a study in which a single heavy chain specific for a glycoprotein antigen of the human immunodeficiency virus (HIV) was combined with various light chains of different antigenic specificity. All of the hybrid antibodies bound to the HIV glycoprotein antigen, indicating that the heavy chain alone was sufficient to confer specificity. However, one should not conclude that the light chain is largely irrelevant; in some antibody-antigen reactions, the light chain makes the more important contribution. The actual shape of the antigen binding site formed by whatever combination of CDRs are used in a particular antibody has been shown to vary dramatically. As pointed out in Chapter 3, contacts between a large globular protein antigen and antibody occur over a broad, often rather flat, undulating face. In the area of contact, protrusions or depressions on the antigen are likely to match complementary depressions or protrusions on the antibody. In the case of the well studied lysozyme/anti-lysozyme system, crystallographic studies have shown that the surface areas of interaction are quite large, ranging from about 650 Å2 to more than 900 Å2 . Within this area, some 15–22 amino acids in the antibody contact the same number of residues in the protein antigen. In contrast, antibodies bind smaller antigens, such as small haptens, in smaller, recessed pockets in which the ligand is buried. This is nicely illustrated by the interaction of the small octapeptide hormone angiotensin II with the binding site of an anti-angiotensin antibody (Figure 4-10). Conformational Changes May Be Induced by Antigen Binding As more x-ray crystallographic analyses of Fab fragments were completed, it became clear that in some cases binding of antigen induces conformational changes in the antibody, antigen, or both. Formation of the complex between neuraminidase and anti-neuraminidase is accompanied by a change in the orientation of side chains of both the epitope and the antigen-binding site. This conformational change results in a closer fit between the epitope and the antibody’s binding site. In another example, comparison of an anti-hemagglutinin Fab fragment before and after binding to a hemagglutinin peptide antigen has revealed a visible conformational change in the heavy-chain CDR3 loop and in the accessible surface of the binding site. Another striking example of conformational change has been seen in the complex between an Fab fragment derived from a monoclonal antibody against the HIV protease and the peptide epitope of the protease. As shown in Figure 4-11, there are significant changes in the Fab upon binding. In fact, upon antigen binding, the CDR1 region of the light chain moves as much as 1 Å and the heavy chain CDR3 moves 2.7 Å. Thus, in addition to variability in the Antibodies: Structure and Function CHAPTER 4 85 L1 H3 L2 L3 H1 H2 FIGURE 4-11 Structure of a complex between a peptide derived from HIV protease and an Fab fragment from an anti-protease antibody (left) and comparison of the Fab structure before and after peptide binding (right). In the right panel, the red line shows the structure of the Fab fragment before it binds the peptide and the blue line shows its structure when bound. There are significant conformational changes in the CDRs of the Fab on binding the antigen. These are especially pronounced in the light chain CDR1 (L1) and the heavy chain CDR3 (H3). [From J. Lescar et al., 1997, J. Mol. Biol. 267:1207; courtesy of G. Bentley, Institute Pasteur.] 8536d_ch04_076-104 9/5/02 6:19 AM Page 85 mac76 mac76:385 Goldsby et al./Immunology5e: