It has been shown that Tyr residues are unusually localized in the regions of antibodies responsible for contact with antigens (Padlan, E. A. (1990) Proteins Struct. Funct. Genet. 7, 112-124). In order to clarify the role of these Tyr residues in antigen binding, the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, whose structure has been well studied in complex with its antigen, was investigated. Four Tyr residues in the VH chain (HTyr-33, HTyr-50, HTyr-53, and HTyr-58) were replaced with Ala, Leu, Phe, or Trp, and the interactions between these mutant Fv fragments and HEL were studied by inhibition assay of the enzymatic activity of HEL and isothermal titration calorimetry. Twelve mutant Fv fragments could be expressed, but two mutants (HY50W and HY58W) could not be obtained in the Escherichia coli expression system, and a further two mutants (HY33A and HY50A) could not be purified by affinity chromatography. It was shown by inhibition assay that Tyr residues at each mutated site made positive contributions to the interaction to different degrees. Thermodynamic studies showed that the role of Tyr residues in antigen binding was to obtain enthalpic energy. The roles of Tyr residues in antibody HyHEL10 for the association with antigen, HEL, can be summarized as follows: 1) formation of hydrogen bonds by the hydroxyl group, 2), creating more favorable interactions through the aromatic ring and decreasing the entropic loss upon binding, and 3) allowing hydrophobic interaction through the side chain. The four Tyr residues studied here were found to play significant roles in the association in various ways.

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.A major goal of molecular biology is to describe biological phenomena in terms of interaction between molecules. Recently, thanks to progress in structural biology, several quaternary structures of biomolecular complexes have been clarified, and now a number of biological events can be described precisely at the atomic level. Among them, DNA-repressor (1), enzyme-inhibitor (2, 3) and antigen-antibody interactions (4, 5) have been most extensively investigated at the atomic level.Antigen-antibody interactions have been studied in detail not only from an immunological but also a biochemical viewpoint as a model of protein-ligand interaction. The most striking feature of antigen-antibody interactions is the creation of strict specificity and high affinity of antibodies for their antigens. It is known that unique conformations and special locations on the surfaces of proteinaceous antigens are recognized by antibodies (6). Among the amino acids located on protein surfaces, it is believed that antibodies recognize charged residues by forming (1) hydrogen bonds and (2) salt bridges and that salt bridge plays a significant role in the interaction (7-9).For detailed elucidation of antigen-antibody interactions, a number of approaches have been attempted, including structural analysis (10 -13) and calculation and modeling (14 -16). However, thermodynamic analysis of antigen-antibody interaction for analyzing precisely the contribution of noncovalent forces to the interaction is most suitable (17)(18)(19)(20)(21)(22), especially when combined with site-directed mutagenesis of structurally well defined ones (23-25). We have focused on the interaction of hen egg white lysozym...

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A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.

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