8536d_cho7 161-184 8/16/02 12: 09 PM Page 166 mac100 mac 100: 1?/8_tm: 8536d: Goldsby et al./Immunology Se 166 PART I1 Generation of B-Cell and T-Cell Response between strains that differ in only a few genes within the Class I Molecules Have a Glycoprotein Heavy MHC. Furthermore, the generation of new H-2 haplotypes Chain and a Small Protein Light Chain under the experimental conditions of congenic strain devel opment provides an excellent illustration of the means by Class I MHC molecules contain a 45-kilodalton(kDa)a which the MHC continues to maintain heterogeneity even in chain associated noncovalently with a 12-kDa B2-microglob populations with limited diversity. brane glycoprotein encoded by polymorphic genes within the A,B, and C regions of the human HLA complex and within the K and D/L regions of the mouse H-2 complex(see Figure MHC Molecules and Genes 7-1). B2-Microglobulin is a protein encoded by a highly con- served gene located on a different chromosome. Association Class I and class II MHC molecules are membrane-bound of the a chain with B2-microglobulin is required for expres- glycoproteins that are closely related in both structure and sion of class I molecules on cell membranes. The a chain is function. Both class I and class II MHC molecules have been anchored in the plasma membrane by its hydrophobic trans isolated and purified and the three-dimensional structures membrane segment and hydrophilic cytoplasmic tail. of their extracellular domains have been determined by x Structural analyses have revealed that the a chain of class I ay crystallography. Both types of membrane glycoproteins MHC molecules is organized into three external domains function as highly specialized antigen-presenting molecules (al, a2, and a3), each containing approximately 90 amino that form unusually stable complexes with antigenic pep- acids; a transmembrane domain of about 25 hydrophobic tides, displaying them on the cell surface for recognition by amino acids followed by a short stretch of charged(hy T cells. In contrast, class III MHC molecules are a group of drophilic)amino acids; and a cytoplasmic anchor segment of unrelated proteins that do not share structural similarity 30 amino acids. The B2-microglobulin is similar in size and and common function with class I and II molecules. The organization to the a3 domain; it does not contain a trans- class lll molecules will be examined in more detail in later membrane region and is noncovalently bound to the class I chapters glycoprotein Sequence data reveal homology between the a3 Class i molecule Class ll molecule membrane. distal Membrane-proximal a3 β2 microglobulin a2 β2 domains (g-fold structure) Transmembrane segment Cytoplasmic tail FIGURE7-5Schematic diagrams of a class I and a class ll MHc membrane-proximal domains possess the basic immunoglobulin- molecule showing the external domains, transmembrane segment, fold structure; thus, class l and class ll MHC molecules are classified and cytoplasmic tail. The peptide-binding cleft is formed by the mem. as members of the immunoglobulin superfamily. brane- distal domains in both dass i and class ll molecules. The
between strains that differ in only a few genes within the MHC. Furthermore, the generation of new H-2 haplotypes under the experimental conditions of congenic strain development provides an excellent illustration of the means by which the MHC continues to maintain heterogeneity even in populations with limited diversity. MHC Molecules and Genes Class I and class II MHC molecules are membrane-bound glycoproteins that are closely related in both structure and function. Both class I and class II MHC molecules have been isolated and purified and the three-dimensional structures of their extracellular domains have been determined by xray crystallography. Both types of membrane glycoproteins function as highly specialized antigen-presenting molecules that form unusually stable complexes with antigenic peptides, displaying them on the cell surface for recognition by T cells. In contrast, class III MHC molecules are a group of unrelated proteins that do not share structural similarity and common function with class I and II molecules. The class III molecules will be examined in more detail in later chapters. Class I Molecules Have a Glycoprotein Heavy Chain and a Small Protein Light Chain Class I MHC molecules contain a 45-kilodalton (kDa) chain associated noncovalently with a 12-kDa 2-microglobulin molecule (see Figure 7-5). The chain is a transmembrane glycoprotein encoded by polymorphic genes within the A, B, and C regions of the human HLA complex and within the K and D/L regions of the mouse H-2 complex (see Figure 7-1). 2-Microglobulin is a protein encoded by a highly conserved gene located on a different chromosome. Association of the chain with 2-microglobulin is required for expression of class I molecules on cell membranes. The chain is anchored in the plasma membrane by its hydrophobic transmembrane segment and hydrophilic cytoplasmic tail. Structural analyses have revealed that the chain of class I MHC molecules is organized into three external domains (1, 2, and 3), each containing approximately 90 amino acids; a transmembrane domain of about 25 hydrophobic amino acids followed by a short stretch of charged (hydrophilic) amino acids; and a cytoplasmic anchor segment of 30 amino acids. The 2-microglobulin is similar in size and organization to the 3 domain; it does not contain a transmembrane region and is noncovalently bound to the class I glycoprotein. Sequence data reveal homology between the 3 166 PART II Generation of B-Cell and T-Cell Responses α1 α2 β1 β2-microglobulin β2 Transmembrane segment Cytoplasmic tail α2 α1 α3 S Class I molecule Class II molecule S S S S S S S S S S S Peptide-binding cleft Membrane-distal domains Membrane-proximal domains (Ig-fold structure) FIGURE 7-5 Schematic diagrams of a class I and a class II MHC molecule showing the external domains, transmembrane segment, and cytoplasmic tail. The peptide-binding cleft is formed by the membrane-distal domains in both class I and class II molecules. The membrane-proximal domains possess the basic immunoglobulinfold structure; thus, class I and class II MHC molecules are classified as members of the immunoglobulin superfamily. 8536d_ch07_161-184 8/16/02 12:09 PM Page 166 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-:
8536d_cho7 161-184 8/16/02 12: 09 PM Page 167 mac100 mac 100: 1?/8_tm: 8536d: Goldsby et al./Immunology Se Major Histocompatibility Complex CHAPTER 7 Peptide-binding cleft a1 domain a 2 domain Sheets a2 domain g a3 domain FIGURE7-6Representations of the three-dimensional structure of munoglobulin- fold structure of the a3 domain and B2-microglobulin the external domains of a human class I MHC molecule based on x. (b) The al and a2 domains as viewed from the top, showing the ray crystallographic analysis. (a) Side view in which the B strands are peptide-binding cleft consisting of a base of antiparallel p strands depicted as thick arrows and the a helices as spiral ribbons. Disulfide and sides of a helices. this cleft in class I molecules can accommo- bonds are shown as two interconnected spheres. The al and a2 do- date peptides containing 8-10 residues ding cleft. Note the im- domain,B2-microglobulin, and the constant-region domains which is not surprising given the considerable sequence sim- in immunoglobulins. The enzyme papain cleaves the a chain ilarity with the immunoglobulin constant regions, class I just 13 residues proximal to its transmembrane domain, re- MHC molecules and B2-microglobulin are classified as leasing the extracellular portion of the molecule, consisting of members of the immunoglobulin superfamily( see Figure al,a2, a3, and B2-microglobulin. Purification and crystal- 4-20). The a3 domain appears to be highly conserved among lization of the extracellular portion revealed two pairs of in- class I MHC molecules and contains a sequence that interacts teracting domains: a membrane-distal pair made up of the al with the CD8 membrane molecule present on Tc cells and a2 domains and a membrane-proximal pair composed of B2-Microglobulin interacts extensively with the a3 do- the a3 domain and B2-microglobulin( Figure 7-6a) main and also interacts with amino acids of the al and a2 The al and o2 domains interact to form a platform of domains. The interaction of B2-microglobulin and a peptide eight antiparallel B strands spanned by two long a-helical re- with a class I a chain is essential for the class I molecule to gions. The structure forms a deep groove, or cleft, approxi- reach its fully folded conformation As described in detail in mately 25 A X 10 A X 11 A, with the long a helices as sides Chapter 8, assembly of class I molecules is believed to occur nd the B strands of the B sheet as the bottom( Figure 7-6b). by the initial interaction of B2-microglobulin with the fold This peptide-binding cleft is located on the top surface of the ing class I a chain. This metastable"empty dimer is then sta- class I MHC molecule, and it is large enough to bind a peptide bilized by the binding of an appropriate peptide to form the of 8-10 amino acids. The great surprise in the x-ray crystallo- native trimeric class I structure consisting of the class I a graphic analysis of class I molecules was the finding of small chain, B2-microglobulin, and a peptide. This complete mole peptides in the cleft that had cocrystallized with the protein. cular complex is ultimately transported to the cell surface These peptides are, in fact, processed antigen and self-pep In the absence of B2-microglobulin, the class I MHC tides bound to the al and a2 domains in this deep groove. chain is not expressed on the cell membrane. This is illus The a3 domain and B2-microglobulin are organized into trated by Daudi tumor cells, which are unable to synthesize two p pleated sheets each formed by antiparallel p strands of B2-microglobulin. These tumor cells produce class I MHCa amino acids. As described in Chapter 4, this structure, known chains, but do not express them on the membrane. However, as the immunoglobulin fold, is characteristic of im- if Daudi cells are transfected with a functional gene encoding munoglobulin domains. Because of this structural similarity, B2-microglobulin, class I molecules appear on the membrane
domain, 2-microglobulin, and the constant-region domains in immunoglobulins. The enzyme papain cleaves the chain just 13 residues proximal to its transmembrane domain, releasing the extracellular portion of the molecule, consisting of 1, 2, 3, and 2-microglobulin. Purification and crystallization of the extracellular portion revealed two pairs of interacting domains: a membrane-distal pair made up of the 1 and 2 domains and a membrane-proximal pair composed of the 3 domain and 2-microglobulin (Figure 7-6a). The 1 and 2 domains interact to form a platform of eight antiparallel strands spanned by two long -helical regions. The structure forms a deep groove, or cleft, approximately 25 Å 10 Å 11 Å, with the long helices as sides and the strands of the sheet as the bottom (Figure 7-6b). This peptide-binding cleft is located on the top surface of the class I MHC molecule, and it is large enough to bind a peptide of 8–10 amino acids. The great surprise in the x-ray crystallographic analysis of class I molecules was the finding of small peptides in the cleft that had cocrystallized with the protein. These peptides are, in fact, processed antigen and self-peptides bound to the 1 and 2 domains in this deep groove. The 3 domain and 2-microglobulin are organized into two pleated sheets each formed by antiparallel strands of amino acids. As described in Chapter 4, this structure, known as the immunoglobulin fold, is characteristic of immunoglobulin domains. Because of this structural similarity, which is not surprising given the considerable sequence similarity with the immunoglobulin constant regions, class I MHC molecules and 2-microglobulin are classified as members of the immunoglobulin superfamily (see Figure 4-20). The 3 domain appears to be highly conserved among class I MHC molecules and contains a sequence that interacts with the CD8 membrane molecule present on TC cells. 2-Microglobulin interacts extensively with the 3 domain and also interacts with amino acids of the 1 and 2 domains. The interaction of 2-microglobulin and a peptide with a class I chain is essential for the class I molecule to reach its fully folded conformation. As described in detail in Chapter 8, assembly of class I molecules is believed to occur by the initial interaction of 2-microglobulin with the folding class I chain. This metastable “empty” dimer is then stabilized by the binding of an appropriate peptide to form the native trimeric class I structure consisting of the class I chain, 2-microglobulin, and a peptide. This complete molecular complex is ultimately transported to the cell surface. In the absence of 2-microglobulin, the class I MHC chain is not expressed on the cell membrane. This is illustrated by Daudi tumor cells, which are unable to synthesize 2-microglobulin. These tumor cells produce class I MHC chains, but do not express them on the membrane. However, if Daudi cells are transfected with a functional gene encoding 2-microglobulin, class I molecules appear on the membrane. Major Histocompatibility Complex CHAPTER 7 167 (b) α1 domain α2 domain α3 domain α2 domain α1 domain β2-microglobulin α helix β sheets (a) Peptide-binding cleft FIGURE 7-6 Representations of the three-dimensional structure of the external domains of a human class I MHC molecule based on xray crystallographic analysis. (a) Side view in which the strands are depicted as thick arrows and the helices as spiral ribbons. Disulfide bonds are shown as two interconnected spheres. The 1 and 2 domains interact to form the peptide-binding cleft. Note the immunoglobulin-fold structure of the 3 domain and 2-microglobulin. (b) The 1 and 2 domains as viewed from the top, showing the peptide-binding cleft consisting of a base of antiparallel strands and sides of helices. This cleft in class I molecules can accommodate peptides containing 8–10 residues. 8536d_ch07_161-184 8/16/02 12:09 PM Page 167 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-:
8536d ch07161-184 8/16/02 12: 09 PM Page 168 mac100 mac 100: 1258 tm: 8536d: Goldsby et al. Immunology 5e 168 PART I1 Generation of B-Cell and T-Cell Response Class ll molecules have two nonidentical Glycoprotein Chains Class II MHC molecules contain two different polypeptide hains, a 33-kDa a chain and a 28-kDa B chain, which asso ciate by noncovalent interactions(see Figure 7-5b) Like class I a chains, class II MHC molecules are membrane-bound glycoproteins that contain external domains, a transmem- brane segment, and a cytoplasmic anchor segment. Each chain in a class ii molecule contains two external domains. I and a2 domains in one chain and Bl and B2 domains in the other. The membrane-proximal a2 and B2 domains, like the membrane-proximal a3/B2-microglobulin domains of class I MHC molecules, bear sequence similarity to the im- munoglobulin-fold structure; for this reason, class II MHC (b) molecules also are classified in the immunoglobulin super- family. The membrane-distal portion of a class II molecule is composed of the al and Bl domains and forms the antigen binding cleft for processed antigen X-ray crystallographic analysis reveals the similarity of lass II and class I molecules, strikingly apparent when the molecules are surperimposed( Figure 7-7). The peptide binding cleft of HLA-DRl, like that in class I molecules, is composed of a floor of eight antiparallel B strands and side of antiparallel a helices. However, the class Il molecule lacks the conserved residues that bind to the terminal residues of short peptides and forms instead an open pocket; class I pre sents more of a socket, class ll an open-ended groove. Thas functional consequences of these differences fine structure will be explored beloy An unexpected difference between crystallized class I and class li molecules was observed for human dri in that the FIGURE7-8Antigen-binding cleft of dimeric class ll DR1 molecule in(a) top view and (b) side view. This molecule crystallized as a dimer of the ap heterodimer. The crystallized dimer is shown with one dri molecule in red and the other dri molecule in blue. the bound peptides are yellow. The two peptide- binding clefts in the dimeric molecule face in opposite directions. From H Brown et al 1993. Nature364:33J latter occurred as a dimer of aB heterodimers, a" dimer of dimers"(Figure 7-8). The dimer is oriented so that the two peptide-binding clefts face in opposite directions. While it has not yet been determined whether this dimeric form exists in vivo, the presence of CD4 binding sites on opposite sides of the class Il molecule suggests that it does. These two sites on the a2 and B2 domains are adjacent in the dimer form and a CD4 molecule binding to them may stabilize class ll dimers The Exon/Intron Arrangement of Class I and FIGURE 7.7 The membrane-distal, peptide-binding cleft of a hu. ll Genes Reflects Their Domain Structure man class Il MHC molecule, HLA-DR1 (blue perimposed over Separate exons encode each region of the class I and ll pro the corresponding regions of a human class I MHC molecule, HLA- teins( Figure 7-9). Each of the mouse and human class I A2(red). From/ H. Brown et aL., 1993, Nature 364: 33. J genes has a 5' leader exon encoding a short signal peptide
Class II Molecules Have Two Nonidentical Glycoprotein Chains Class II MHC molecules contain two different polypeptide chains, a 33-kDa chain and a 28-kDa chain, which associate by noncovalent interactions (see Figure 7-5b). Like class I chains, class II MHC molecules are membrane-bound glycoproteins that contain external domains, a transmembrane segment, and a cytoplasmic anchor segment. Each chain in a class II molecule contains two external domains: 1 and 2 domains in one chain and 1 and 2 domains in the other. The membrane-proximal 2 and 2 domains, like the membrane-proximal 3/2-microglobulin domains of class I MHC molecules, bear sequence similarity to the immunoglobulin-fold structure; for this reason, class II MHC molecules also are classified in the immunoglobulin superfamily. The membrane-distal portion of a class II molecule is composed of the 1 and 1 domains and forms the antigenbinding cleft for processed antigen. X-ray crystallographic analysis reveals the similarity of class II and class I molecules, strikingly apparent when the molecules are surperimposed (Figure 7-7). The peptidebinding cleft of HLA-DR1, like that in class I molecules, is composed of a floor of eight antiparallel strands and sides of antiparallel helices. However, the class II molecule lacks the conserved residues that bind to the terminal residues of short peptides and forms instead an open pocket; class I presents more of a socket, class II an open-ended groove. These functional consequences of these differences in fine structure will be explored below. An unexpected difference between crystallized class I and class II molecules was observed for human DR1 in that the latter occurred as a dimer of heterodimers, a “dimer of dimers” (Figure 7-8). The dimer is oriented so that the two peptide-binding clefts face in opposite directions. While it has not yet been determined whether this dimeric form exists in vivo, the presence of CD4 binding sites on opposite sides of the class II molecule suggests that it does. These two sites on the 2 and 2 domains are adjacent in the dimer form and a CD4 molecule binding to them may stabilize class II dimers. The Exon/Intron Arrangement of Class I and II Genes Reflects Their Domain Structure Separate exons encode each region of the class I and II proteins (Figure 7-9). Each of the mouse and human class I genes has a 5 leader exon encoding a short signal peptide 168 PART II Generation of B-Cell and T-Cell Responses FIGURE 7-7 The membrane-distal, peptide-binding cleft of a human class II MHC molecule, HLA-DR1 (blue), superimposed over the corresponding regions of a human class I MHC molecule, HLAA2 (red). [From J. H. Brown et al., 1993, Nature 364:33.] (a) (b) FIGURE 7-8 Antigen-binding cleft of dimeric class II DR1 molecule in (a) top view and (b) side view. This molecule crystallized as a dimer of the heterodimer. The crystallized dimer is shown with one DR1 molecule in red and the other DR1 molecule in blue. The bound peptides are yellow. The two peptide-binding clefts in the dimeric molecule face in opposite directions. [From J. H. Brown et al., 1993, Nature 364:33.] 8536d_ch07_161-184 8/16/02 12:09 PM Page 168 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: