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.
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.
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.
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.
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.