Antibodyantigen complexes

The introduction of cell hybridization techniques leading to the development of immortalized lymphocytic cell lines (hybridomas) secreting antibodies of predefined specificity allowed the production of monoclonal antibodies (mAbs) in large quantities. These made it possible to study specific antigen-antibody reactions in greater detail, eventually providing a deeper understanding of antigenic determinants and combining site structure. Moreover, the elucidation of the antigen-binding site has been greatly facilitated by the production of antibody variable domain fragments (Fv). As the volume of Fv fragments is half that of the Fab, Fv fragments generally crystallize in smaller asymmetric units and thus diffract to higher resolution.

By 1996 more than 50 structures of antibody fragments, free and complexed with antigen, had been determined by X-ray diffraction techniques. By far the most widely studied antibodies have been of mouse origin. The mAbs were obtained after secondary responses and can be assumed to be highly specific -that is, they recognize only one antigen (and related, cross-reacting antigens sharing the antigenic determinant). Thus, their combining sites are uniquely defined by stereospecific contacts with the antigens. Several BALB/c mouse mAbs have now been studied by X-ray diffraction, including six which form complexes with hen egg-white lysozyme (HEL), two with influenza virus neuraminidase, one with HPr (a histi-dine-containing phosphocarrier protein), one with NC10 staphylococcal nuclease and four with anti-idiotopic antibodies. These results can be correlated with those of other crystallographic, immunochemical and sequence studies related to antigen and hapten recognition by specific antibodies. In most of the complexes above, the combining site is formed by amino acid residues from each of the CDRs of the L and H chains, although the H chain may in several cases contribute more contacts than the L chain. About 15 residues, mostly from the CDRs but including also one or two from the framework regions, are in contact with a similar number from the antigen. The general topology of the site is not that of a big cavity, but rather flat with interspersed depressions and protuberances that closely complement the surface topology of the antigenic determinant.

The nature of the chemical contacts made by the combining site includes charge bridges between ion pairs, hydrogen bonds and van der Waals interactions. If hydrogen bonds are inaccessible to solvent they should contribute appreciably to the stability of antigen-antibody interactions. In the complex of mAbs HyHEL-5 and D44.1 with HEL, two glutamic acid residues of the combining site interact closely with two arginines of the antigen. The crystal structure of HyHEL-5 complexed with a site-directed mutant of HEL (Arg68—»Lys) demonstrated the specificity of electrostatic interactions. While generally considered a conservative mutation, the HEL mutant binds to HyHEL-5 with a 10'-fold reduction in affinity. The crystal structure demonstrated that lysine is unable to form both salt bridges to a HyHEL-5 glutamate, therefore the decrease in affinity of the HyHEL-5 reaction with the mutant HEL was ascribed to the loss of one hydrogen bond. Further evidence of the electrostatic complementarity defining antibody-antigen interactions came from the crystal structure of D1.3 complexed with turkey egg-white lysozyme (TEL). In this case, an important residue in the antibody-anti-gen interface (HEL Glnl21) is replaced by a histidine. Glnl21 makes two hydrogen bonds to carbonyl oxygen and amide nitrogen main-chain atoms of D 1.3 V, CDR-3 (Oel-Ser93 N; Ne2-Phe 91 O). The orien tation of TEL Hisl21 is such that its Ne2 atom is positionally equivalent to the Oel atom of HEL Glnl21 and therefore unable to form a hydrogen bond to the amide nitrogen of D 1.3 Ser93. D1.3 was observed to compensate for this difference in the electrostatic character of HEL121 by undergoing a flip of the main-chain peptide orientation such that the carbonyl oxygen of Trp92 is exposed at the interface, allowing formation of a hydrogen bond to TEL Hisl21 Ne2 (Figure 1).

Since hydrogen bonds and van der Waals interactions require a somewhat strict complementarity, they can be taken as the basis of antigen-antibody specificity. In the combining sites there is a high relative proportion of aromatic residues Tyr and Trp. Given their bulky size, these contribute a maximum of van der Waals contacts as well as affording the possibility of hydrogen bond formation to their polar atoms. The area of contact between antigen and antibody can be defined as that which becomes inaccessible to solvent after the formation of a complex. The surface buried by protein antigens is of about 600 A2 and higher, compatible with the requirements for the formation of a stable complex. This observation is in agreement with that made in other protein-interacting systems in which subunits make contacts through a closely packed interface occupying a large surface area. The crystal structure of a Trp—»Asp mutation of D1.3

Figure 1 Conformational differences in antibody D1.3 VL CDR3 induced by differing antigen side-chains. HEL residue Gln121 makes two main-chain hydrogen bonds; to the carbonyl oxygen of D1.3 VL Phe91 and the amide nitrogen of D1.3 VL Ser93. Replacement of the glutamine by histidine in TEL induces a conformational change in the backbone of VL CDR3 which allows the formation of a hydrogen bond between the histidine and the carbonyl oxygen of VL Trp92. (See also color Plate 8A.)

Figure 1 Conformational differences in antibody D1.3 VL CDR3 induced by differing antigen side-chains. HEL residue Gln121 makes two main-chain hydrogen bonds; to the carbonyl oxygen of D1.3 VL Phe91 and the amide nitrogen of D1.3 VL Ser93. Replacement of the glutamine by histidine in TEL induces a conformational change in the backbone of VL CDR3 which allows the formation of a hydrogen bond between the histidine and the carbonyl oxygen of VL Trp92. (See also color Plate 8A.)

HEL Gln121

His121

HEL Gln121

His121

exemplifies the importance of interacting surface complementarity and van der Waals interactions. The VL Trp92—»Asp mutation results in a 103 reduction in the affinity of D1.3 for HEL. Trp92 makes extensive van der Waals contacts with the antigen. With the substitution of Asp about 150 A2 of surface are lost to the antibody-antigen complex, resulting in the reduction of affinity (Figure 2).

While a few buried water molecules have been reported in other antibody-antigen complexes, the D1.3-HEL complex contains a large number of bound water molecules which bridge the antibody and antigen. This water contributes to the stability of the antibody-antigen complex in two important ways: 1) the network of water molecules contributes to a favorable free energy of complex formation by mediating hydrogen bonds between antibody and antigen; and 2) the water molecules fill cavities in the interface which are destabilizing to complex formation (Figure 3).

No major conformational change seems to take place at the antibody site upon the binding of antigen. Small rearrangements of side-chains and CDR loops have been observed, particularly upon the binding of small antigens, most notably peptides, as well as relative displacements of VH and V^. Thus, antibody-antigen complexes can be viewed as a form of 'lock-and-key' association and small displacements can provide potential diversity in the conformation of the

Figure 2 The effect of a Trp—«Asp mutation on the interaction surface area of the D1,3-HEL complex. A thin slice through the buried surface of the wild-type D1,3-HEL complex (dots) and the mutant D1.3-HEL surface (solid line) demonstrates the loss of interaction surface area in the mutant complex. A 150 A2 loss in surface area accounts for the reduction in affinity of the mutant D1,3-HEL reaction. (See also color Plate 8B.)

Figure 2 The effect of a Trp—«Asp mutation on the interaction surface area of the D1,3-HEL complex. A thin slice through the buried surface of the wild-type D1,3-HEL complex (dots) and the mutant D1.3-HEL surface (solid line) demonstrates the loss of interaction surface area in the mutant complex. A 150 A2 loss in surface area accounts for the reduction in affinity of the mutant D1,3-HEL reaction. (See also color Plate 8B.)

Figure 3 (A) Hydrogen bonding network of the D1,3-HEL interface mediated by bound solvent molecules; 25 water molecules form hydrogen bonds linking the antibody and antigen, directly or through other water molecules. (B) Water molecules in contact with the D1,3-HEL buried surface. Including the 25 bridging water molecules, nearly 50 solvent sites are in contact with the buried surface defined by the D1,3-HEL interface. Many of these water molecules fill internal cavities, further stabilizing the complex. (See also color Plate 8C and D.)

Figure 3 (A) Hydrogen bonding network of the D1,3-HEL interface mediated by bound solvent molecules; 25 water molecules form hydrogen bonds linking the antibody and antigen, directly or through other water molecules. (B) Water molecules in contact with the D1,3-HEL buried surface. Including the 25 bridging water molecules, nearly 50 solvent sites are in contact with the buried surface defined by the D1,3-HEL interface. Many of these water molecules fill internal cavities, further stabilizing the complex. (See also color Plate 8C and D.)

combining site and, perhaps, a mechanism by which the site could adapt to an antigenic determinant without large loss of entropy.

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