Abstract
Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.
Highlights
The specific recognition of ligands by proteins is a fundamental biological phenomenon [1], and the interaction be
We constructed six mutant HyHEL-10 Fv fragments named LN31A, LN31D, LN32A, LN32D, LN92A, and LN92D to elucidate the energetic contributions of direct hydrogen bonds, which are formed by amino acid residues in antibody-antigen interactions
We investigated the interactions between mutant Fv fragments and the hen egg white lysozyme (HEL) antigen by structural and thermodynamic analyses of the resultant complexes
Summary
Activity of HEL by the wild-type Fv fragment and the five of the mutant Fv fragments LN31A, LN31D, LN32A, LN32D, and Expression and Purification of Fv Fragments. To elucidate the role of interfacial asparagine residues in a similar level of inhibition of HEL enzymatic activity, and for direct hydrogen bond formation in HyHEL-10 Fv-HEL com- LN92D this level was only slightly lower than that of the wild plexes, we constructed, expressed, and purified six mutant Fv type. LN31A and LN32A displayed no inhibitory activities obtained purities of greater than 95%, and the final yields were toward HEL. These results suggest that the mutations harbored greater than 10 mg/liter of culture. More, the yield of LN32A was low following purification, despite the level of expression, and only a limited number of experiments were performed for LN32A
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