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Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune system in Health and Disease. 5th edition. Brand-new York: Garland Science; 2001.
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We have explained the framework of the antibody molecule and how the V areas of theheavy and light chains fold and pair to kind the antigen-binding site. In this partof the chapter we will certainly look in ~ the antigen-binding site in an ext detail. Us willdiscuss the various ways in i m sorry antigens deserve to bind come antibody and deal with thequestion of how variation in the sequences of the antibody V domains determines thespecificity because that antigen.
3-6. Localized regions of hypervariable sequence type the antigenbindingsite
The V areas of any given antibody molecule different from those that every other.Sequence variability is not, however, dispersed evenly throughout the Vregions yet is concentrated in specific segments the the V region. Thedistribution of change amino acids can be seen plainly in what is termed avariability plot (Fig. 3.6), in which the amino acidsequences of many different antibody V regions are compared. Three segments ofparticular variability can be figured out in both the VH andVL domains. They are designated hypervariableregions and are denoted HV1, HV2, and HV3. In the light chains theseare around from residues 28 come 35, native 49 come 59, and also from 92 come 103,respectively. The many variable component of the domain is in the HV3 region. Theregions between the hypervariable regions, which make up the remainder of the V domain, display less variability and also are termed the frame regions. There are four such regions in each Vdomain, designated FR1, FR2, FR3, and FR4.
There room discrete regions of hypervariability in Vdomains. A variability plot obtained from compare of the amino acidsequences of numerous dozen heavy-chain and light-chain V domains. Ateach amino acid place the degree of variability is the proportion (more...)
The framework regions kind the β sheets that carry out the structural framework ofthe domain, whereas the hypervariable sequences exchange mail to three loops in ~ theouter sheet of the β barrel, which room juxtaposed in the folded domain (Fig. 3.7). Thus, not only is sequencediversity focused in particular parts of the V domain but it is localizedto a particular region on the surface ar of the molecule. Once the VHand VL domain names are paired in the antibody molecule, the hypervariableloops from every domain are brought together, creating a solitary hypervariablesite at the tip of each arm of the molecule. This is the binding site forantigen, the antigen-binding siteor antibody combining site. Thethree hypervariable loops recognize antigen specificity by forming a surfacecomplementary come the antigen, and are an ext commonly termed thecomplementarity-determining regions, or CDRs (CDR1,CDR2, and CDR3). Due to the fact that CDRs native bothVH and VL domains add to the antigen-bindingsite, the is the mix of the heavy and the irradiate chain, and not eitheralone, the determines the last antigen specificity. Thus, one way in i m sorry theimmune system is may be to create antibodies of various specificities is bygenerating different combinations the heavy- and also light-chain V regions. Thismeans of producing variability is known as combinatorial diversity; we will encounter a second form ofcombinatorial diversity as soon as we take into consideration in thing 4 exactly how the genes encoding the heavy- andlight-chain V regions are produced from smaller segments the DNA.
The hypervariable areas lie in discrete loops the the foldedstructure. When the hypervariable areas (CDRs) room positioned on the structureof a V domain it can be seen that castle lie in loops that space broughttogether in the urgently structure. In the (more...)
3-7. Antibodies bind antigens via contacts v amino acids in CDRs, yet thedetails of binding rely upon the size and also shape of the antigen
In at an early stage investigations the antigen binding come antibodies, the only availablesources of huge quantities the a single kind of antibody molecule to be tumors ofantibody-secreting cells. The antigen specificities that the tumor-derivedantibodies were unknown, so plenty of compounds had to be screened to identifyligands that can be supplied to research antigen binding. In general, the substancesfound to tie to these antibodies were haptens (see section 3-4) such together phosphorylcholine or vitaminK1. Structural analysis of complexes the antibodies through theirhapten ligands noted the first direct proof that the hypervariable regionsform the antigen-binding site, and demonstrated the structural basis ofspecificity for the hapten. Subsequently, with the exploration of techniques ofgenerating monoclonal antibodies (see AppendixI, ar A-12), the became feasible to make large amounts the pureantibodies particular for many different antigens. This has provided a moregeneral snapshot of exactly how antibodies connect with their antigens, confirming andextending the see of antibody-antigen interactions obtained from the research ofhaptens.
The surface ar of the antibody molecule created by the location junxtap of the CDRs ofthe heavy and light chain creates the site to i m sorry an antigen binds. Clearly,as the amino acid sequences the the CDRs are various in different antibodies,so are the forms of the surfaces produced by these CDRs. As a general principle,antibodies bind ligands who surfaces room complementary to the of theantibody. A small antigen, such together a hapten or a quick peptide, normally bindsin a bag or groove lying in between the heavy- and light-chain V domain names (Fig. 3.8, left and center panels). Otherantigens, such as a protein molecule, can be the the very same size as, or largerthan, the antibody molecule itself, and also cannot fit right into a groove or pocket. Inthese cases, the interface in between the 2 molecules is frequently an extendedsurface that involves all of the CDRs and, in part cases, component of the frameworkregion of the antibody (Fig. 3.8, rightpanel). This surface require not be concave however can be flat, undulating, or evenconvex.
Antigens deserve to bind in pockets or grooves, or on expanded surfacesin the binding web page of antibodies. The panels in the peak row present schematic depictions of thedifferent types of binding website in a Fab fragment of one antibody:left, pocket; center, (more...)
3-8. Antibodies bind to conformational forms on the surface of antigens
The biological duty of antibodies is to tie to pathogens and their products,and to facilitate their removal indigenous the body. One antibody generally recognizesonly a small an ar on the surface of a big molecule such together a polysaccharideor protein. The structure well-known by an antibody is called an antigenic determinant or epitope. Several of the many importantpathogens have actually polysaccharide coats, and antibodies that identify epitopesformed by the street subunits of this molecules are crucial in providingimmune defense from together pathogens. In plenty of cases, however, the antigens thatprovoke one immune solution are proteins. For example, security antibodiesagainst viruses identify viral coat proteins. In such cases, the structuresrecognized by the antibody are located on the surface ar of the protein. Such sitesare most likely to be created of amino acids from different parts that the polypeptidechain that have been brought together by protein folding. Antigenic determinantsof this sort are well-known as conformational or discontinuous epitopes since thestructure recognized is composed of segments of the protein that arediscontinuous in the amino acid sequence the the antigen but are brought togetherin the three-dimensional structure. In contrast, one epitope created of a singlesegment of polypeptide chain is termed a continuous or direct epitope. Although mostantibodies raised against intact, fully folded proteins recognize discontinuousepitopes, some will certainly bind peptide fragments of the protein. Conversely,antibodies raised versus peptides of a protein or versus synthetic peptidescorresponding to component of that is sequence are occasionally discovered to bind to thenatural folded protein. This renders it possible, in some cases, to usage syntheticpeptides in vaccines the aim at increasing antibodies versus a pathogenprotein.
3-9. Antigen-antibody interaction involve a selection of forces
The interaction in between an antibody and also its antigen deserve to be disrupted by high saltconcentrations, extremes of pH, detergents, and sometimes by competition withhigh concentration of the pure epitope itself. The binding is therefore areversible noncovalent interaction. The forces, or bonds, affiliated in thesenoncovalent interactions room outlined in Fig.3.9.
The noncovalent forces that host together the antigen:antibodycomplex. Partial charges found in electric dipoles are presented as δ+or δ-. Electrostatic pressures diminish as the inversesquare the the distance separating the charges, whereas (more...)
Electrostatic interaction occur between charged amino acid side chains, together insalt bridges. Interactions also occur in between electric dipoles, together in hydrogenbonds, or have the right to involve short-range van der Waals forces. High salt concentrationsand extremes the pH disrupt antigen-antibody binding through weakening electrostaticinteractions and/or hydrogen bonds. This rule is to work in thepurification of antigens using affinity columns the immobilized antibodies, andvice versa because that antibody purification (see attachment I, section A-5). Hydrophobic interactions occur when twohydrophobic surface come together to exclude, water. The toughness of ahydrophobic interaction is proportional come the surface ar area the is concealed fromwater. For some antigens, hydrophobic interactions most likely account for many ofthe binding energy. In some cases, water molecules space trapped in pockets in theinterface between antigen and antibody. These trapped water molecules might alsocontribute come binding, especially between polar amino acid residues.
The contribution of every of these pressures to the in its entirety interaction relies onthe certain antibody and also antigen involved. A striking distinction betweenantibody interactions v protein antigens and most various other naturalprotein-protein interactions is that antibodies own many fragrant aminoacids in their antigen-binding sites. This amino acids participate greatly invan der Waals and also hydrophobic interactions, and sometimes in hydrogen bonds. Ingeneral, the hydrophobic and van der Waals forces operate over very short rangesand offer to pull together two surface that are complementary in shape: hillson one surface must fit right into valleys top top the other for good binding to occur. Incontrast, electrostatic interactions between charged side chains, and also hydrogenbonds bridging oxygen and/or nitrogen atoms, accommodate certain features orreactive teams while strengthening the interaction overall.
For example, in the complex of hen egg-white lysozyme with the antibody D1.3(Fig. 3.10), solid hydrogen bondsare formed between the antibody and also a certain glutamine in the lysozymemolecule the protrudes in between the VH and VL domains.Lysozymes native partridge and also turkey have one more amino mountain in ar of theglutamine and do no bind come the antibody. In the high-affinity complicated of henegg-white lysozyme with one more antibody, HyHel5 (see Fig. 3.8c), 2 salt bridges in between two simple arginineson the surface ar of the lysozyme interact with 2 glutamic acids, one each fromthe VH CDR1 and CDR2 loops. Again, lysozymes that lack one that the twoarginine residues show a 1000-fold diminish in affinity. Back overallsurface complementarity need to play crucial part in antigen-antibodyinter-actions, certain electrostatic and also hydrogen-bonding interaction appearto identify antibody affinity. In many antibodies that have been studied atthis level the detail, only a few residues make a major contribution to thebinding energy. Genetic design by site-directed mutagenesis can furthertailor one antibody\"s binding to its complementary epitope.
The complex of lysozyme v the antibody D1.3. The interaction of the Fab fragment of D1.3 with hen egg-whitelysozyme is shown, v the lysozyme in blue, the heavy chain inpurple and the irradiate chain in yellow. A glutamine residue oflysozyme, displayed (more...)
X-ray crystallographic analysis of antigen:antibody complexes has demonstratedthat the hypervariable loops (complementarity-determining regions) ofimmunoglobulin V regions identify the specificity the antibodies. V proteinantigens, the antibody molecule contacts the antigen over a wide area of itssurface the is complementary come the surface known on the antigen.Electrostatic interactions, hydrogen bonds, valve der Waals forces, andhydrophobic interactions deserve to all add to binding. Amino mountain side chainsin many or every one of the hypervariable loops make contact with antigen anddetermine both the specificity and the affinity the the interaction. Various other partsof the V an ar play little part in the direct contact with the antigen butprovide a stable structural framework for the hypervariable loops and also helpdetermine your position and conformation. Antitoxin raised versus intactproteins normally bind to the surface ar of the protein and make call withresidues that space discontinuous in the main structure the the molecule; theymay, however, occasionally bind peptide fragments of the protein, and also antibodiesraised versus peptides acquired from a protein have the right to sometimes be provided to detectthe native protein molecule. Peptides binding to antibodies generally bind in thecleft in between the V areas of the heavy and also light chains, where they makespecific contact with some, but not have to all, of the hypervariable loops.This is also the usual setting of binding for carbohydrate antigens and smallmolecules such together haptens.
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