Detailed Structure of Antibodies- Immunoglobulin Domains, CDRs and FRs (Part 2- Antibody Basics)
Welcome to the 2nd part of the 13-part series on Antibody basics. You can read the first part here to understand what are antibodies and their basic structure.
The immunoglobulin structure is determined by the protein’s primary, secondary, tertiary, and quaternary organization. The primary structure of the immunoglobulin refers to the sequence of amino acids in the variable and constant regions of the heavy and light polypeptide chains. The secondary structure of immunoglobulin refers to the folding of the immunoglobulin polypeptide chain back and forth upon itself to form a β pleated sheet. The tertiary structure refers to the folding of the polypeptide chain into compact globular domains. And the globular domains of adjacent heavy and light polypeptide chains interact to form a quaternary structure, which eventually creates the functional immunoglobulin molecule that can specifically interact with the antigen and perform several biological functions.
Immunoglobulin Domains
The heavy and light polypeptide chains contain several homologous units of 110 amino acid residues, and each unit is termed as a domain. An intrachain disulfide bond is present in each domain, forming a loop of about 60 amino acids. In the case of light chains of the antibody, there is one variable domain (VL) and one constant domain (CL), and in the case of the heavy chains, there is one variable domain VH and either 3 or 4 constant domains designated as CH1, CH2, CH3, and CH4.
The overall shape of the immunoglobulin domain can be defined as the sandwich of two β pleated sheets, each containing antiparallel β strands of amino acid residues connected by loops of various lengths.
The β strands within the β pleated sheet are stabilized by hydrogen bonds that connect the amino group in one strand with the carbonyl group of an adjacent strand. The two β pleated sheets are stabilized by hydrophobic interactions between them and by the conserved disulfide bond. An analogy of the structure of the immunoglobulin domain can be made to two slices of bread, butter between them, and a toothpick holding the slices together. The two slices of bread are β pleated sheets; butter represents hydrophobic interactions, and toothpick represents the intrachain disulfide bond.
The variable (V) and constant © domains of immunoglobulins differ in the number and regularity of the β strands forming the β pleated sheets. For instance, the constant domain of the light chain is built up from seven β strands arranged such that four strands form one β sheet and three strands form the second β sheet. In contrast, the V domains contain nine β strands instead of seven, four strands form one β sheet, and five strands form the second sheet.
Non-covalent interactions form links between the identical domains of heavy chains and between non-identical domains of heavy and light chains.
Complementarity determining regions (CDRs)
The specific regions within the variable region in each light and heavy chain responsible for generating the antigen-binding site of the antibody are termed complementarity determining regions abbreviated as CDRs. As already discussed, the variable regions in the light and heavy chains form the antigen-binding site. There are two antigen-binding sites in an antibody molecule. Also, the heavy and light polypeptide chains contain several homologous units of 110 amino acid residues. Each unit is termed a domain, which is a sandwich of two β pleated sheets, each sheet containing antiparallel β strands of amino acid residues connected by loops of varied lengths.
The exciting thing is that the maximum variation in the variable domain of heavy and light chains is present in the amino acid sequences of loops that join the β strands. These loop regions are called hypervariable regions.
The light and heavy variable domains fold in a manner that brings the hypervariable regions of both chains together to create the antigen-binding site or paratope.
The surface of the antigen-binding site formed by the hypervariable regions is complementary to the structure of epitopes to generate antigen specificity. An epitope is the specific part of an antigen to which the antibody binds. Since the hypervariable regions are complementary to the structure of epitopes, they are called complementarity determining regions or CDRs.
Within the variable domain of each heavy and light chain, there are 3 CDRs. The antigen-binding site is formed by pairing the variable domain of one light chain with the variable domain of one heavy chain.
Each domain contributes to three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3, in the light chain variable region and CDR-H1, CDR-H2, and CDR-H3 in the variable region of the heavy chain. Since the CDRs form the antigen-binding site of an antibody molecule, thus a total of 6 CDRs constitute one antigen-binding site. In addition, because CDRs from both VH and VL domains contribute to the antigen-binding site, it is the combination of the heavy and the light chain, and not either alone, that determines the final antigen specificity.
Similarly, the second antigen-binding site is formed by pairing variable domains of the other light chain and heavy chain. Thus, the second antigen-binding site will also be created by another 6 CDRs.
Therefore, a single antibody molecule contains a total of 12 CDRs.
The antigen specificity of an antibody determines its ability to distinguish the subtle differences among antigens. Since the CDRs are the antigen-binding sites, they allow the antibody to bind to the specific antigen only and account for the diversity of antigens that a repertoire of antibodies can recognize.
Framework region
Variable domains of both heavy and light chains can be divided into two regions: complementarity determining regions that have been discussed and framework regions abbreviated as FRs. Unlike the complementarity determining regions (CDRs) which show maximum variation in the amino acid sequences, framework regions are conserved regions of variable regions and show less variation in the amino acid sequences. These regions support the binding of CDRs to the antigen. In other words, the framework regions serve as a scaffold to hold the CDRs in position to contact antigen so that the CDRs can take the correct orientation and position to bind to the antigen. The framework regions are present in the variable domains of both heavy and light chains.
The CDRs are present on the loops that join the β strands in the variable domains of both heavy and light chains. In contrast, the β-sheet structure is a framework region that serves as a scaffold to hold the CDRs in position to contact antigen.
Binding of antigen to CDRs
As already discussed, the CDRs generate the antigen-binding site of the antibody, and the antigen-binding site of an antibody is complementary to the structure of epitopes to generate antigen specificity. Clearly, as the amino acid sequences of the CDRs are different in different antibodies, so are the shapes of the surfaces created by these CDRs; thus, different antibodies bind with different antigens. Therefore, as a general principle, antibodies bind antigens whose surfaces are complementary to that of the antibody.
The antigen can bind to the antigen-binding site formed by CDRs in 3 possible ways.
· Suppose the antigen is a large globular protein, for instance, viral coat proteins or polysaccharide coats of pathogens; in that case, the contact between this antigen and the antibody occurs over a broad, flat, and undulating surface created by the CDRs. In the area of contact, protrusions or depressions present on the antigen are likely to match complementary depressions or protrusions present on the antibody molecule. In this scenario, around 15–22 amino acids in the CDRs of antibody contact the same number of residues in the protein antigen.
· In smaller antigens such as small haptens, antigen-antibody interaction occurs in narrow and deep pockets created by the CDRs.
· In some cases, antigen binding to the antibody induces a conformational change in antibody, antigen, or both. This conformational change results in a closer fit between the epitope and antigen-binding site of the antibody.
Further X-ray studies have confirmed that more residues in the heavy-chain CDRs appear to contact antigen than in the light-chain CDRs. Thus the variable domain of the heavy chain often contributes more than the variable domain of the light chain. However, it is not the conclusion that the light chain is irrelevant because the antigen-binding site is only formed when the light and heavy variable domains fold in a manner that brings the CDRs of both chains together to create the Ag binding site or paratope.
Thus, the combination of the heavy and the light chain, not alone, determines the final antigen specificity. Even in some antigen-antibody interactions, studies have shown that the light chain makes the more significant contribution.
Hinge Region
Out of the five heavy chains, 𝛾, δ and α heavy chains contain an extended amino acid sequence between the CH1 and CH2 domains that has no homology with the other domains. This region is called the hinge region.
Hinge is a flexible amino acid stretch that provides flexibility to IgG, IgD, and IgA and allows independent movement of the Fab fragments. As a result, the two Fab arms can move relative to each other, leading to better interaction with the antigen. Proline and cysteine are the two prominent amino acids present in the hinge region.
A large number of proline residues in the hinge region makes the antibody vulnerable to getting cleaved by proteolytic enzymes such as papain or pepsin. On the other hand, the cysteine residues present in the hinge region form interchain disulfide bonds, enabling the two heavy chains to bind together.
The number of interchain disulfide bonds in the hinge region varies significantly among different classes of antibodies.
µ and ε heavy chains lack a hinge region, but they have an additional domain of 110 amino acids with hinge-like features.
Thus, there are four constant domains in IgM and IgE Abs because of the absence of hinge region, whereas there are three constant domains in IgG, IgD, and IgA antibodies because of the presence of the hinge region.
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