Role of salt bridge formation in antigen-antibody interaction. Entropic contribution to the complex between hen egg white lysozyme and its monoclonal antibody HyHEL10.
For elucidation of the role of salt bridge formation in the antigen-antibody complex, the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, the structure of which has been well characterized and forms one salt bridge (Lys97 of HEL and Asp32 of HyHEL10 heavy chain variable region (VH)), was investigated. Asp32 of VH was substituted with Ala, Asn, or Glu by site-directed mutagenesis, and the interaction between HEL and the mutant fragments of the variable region of light chain was investigated by inhibition of the enzymatic activity of HEL and isothermal titration calorimetry. Inhibition assay indicated that these mutations lowered the inhibition only slightly. Thermodynamic study indicated that the negative enthalpic change in the interaction between each of the mutant variable regions of light chain and HEL was significantly increased, although the association constant was slightly decreased, suggesting that these mutations increased the entropy change upon antigen-antibody binding. These results indicate that the role of salt bridge formation in the HyHEL10-HEL interaction is to lower the entropic loss due to binding. In the mutant proteins, the numbers of residues that were perturbed structurally on binding increased, suggesting that the salt bridge suppresses excess structural movement of the antibody upon binding.
Tsumoto
K
K
Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-77, Japan.
Ogasahara
K
K
Ueda
Y
Y
Watanabe
K
K
Yutani
K
K
Kumagai
I
I
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D002151:Calorimetry; D002645:Chickens; D019277:Entropy; D007128:Immunoglobulin Fragments / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry / Q000276:immunology*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins; D012492:Salts
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D012492:Salts; C055966:immunoglobulin Fv; D009113:Muramidase