©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Tyr Residues in the Contact Region of Anti-lysozyme Monoclonal Antibody HyHEL10 for Antigen Binding (*)

(Received for publication, February 13, 1995; and in revised form, May 16, 1995)

Kouhei Tsumoto (§) Kyoko Ogasahara (1) Yoshitaka Ueda Kimitsuna Watanabe Katsuhide Yutani (1) Izumi Kumagai (§)

From the Department of Chemistry and Biotechnology, Faculty of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan and the Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Antibodies play a significant role in the immune system, which acts to recognize and eliminate foreign molecules. Antigen-antibody interactions have been studied extensively from various standpoints including immunology, biochemistry, and structural biology (Davies et al., 1990). Proteinaceous antigen-antibody interaction is one of the best models for studying protein-protein interaction (Janin and Chothia, 1990; Webster et al., 1994) and has provided considerable knowledge about the general rules of protein-protein interactions.

Antibodies can recognize foreign molecules by varying their antigen binding regions supported by framework regions called immunoglobulin folds. The antigen binding sites of antibodies are composed of six hypervariable loops, which are often called complementarity determining regions (CDR) (^1)(Kabat et al., 1991). Over the last few years, detailed studies of antibodies have revealed that the amino acid compositions of CDR are biased (Padlan, 1990; Mian et al., 1991). In particular, Tyr residues are found to be especially localized in CDR and seem to be widely used for the binding of antigens such as hapten (Satow et al., 1986; Herron et al., 1989) sugar (Cygler et al., 1991), DNA (Cygler et al., 1987), and protein (Amit et al., 1986; Colman et al., 1987; Padlan et al., 1989; Chitarra et al., 1993). The ratio of the area of Tyr residues in contact regions of CDR to total contact area is reported to be relatively high (Padlan, 1990). A biased localization of aromatic rings, particularly Tyr residues in CDR, has been reported to be a characteristic of antibodies and might be a biologically significant feature of antigen-antibody interactions (Mian et al., 1991). On the other hand, structural study of other protein-protein interactions and protein-ligand interactions (for instance, hormone-receptor interaction and protease-inhibitor interaction) has shown no particularly prevalent amino acid composition in the contact regions of these proteins (Janin and Chothia, 1990). Therefore, studying the role of Tyr residues in antigen binding would make a significant contribution to understanding the fundamental mechanism of antigen-antibody interactions.

For accurate description of protein-protein interaction, both structural and thermodynamic analyses are necessary. Especially, the contribution of noncovalent bonds such as electrostatic and hydrophobic interaction to protein-protein interactions can be precisely described by thermodynamic studies. Recently, several groups have studied protein-ligand interactions using isothermal titration calorimetry (Wiseman, et al., 1989; Varadarajan et al., 1992; Ogasahara et al., 1992), and isothermal titration calorimetry has now become one of the most powerful methods for precise analysis of the thermodynamics of biochemical interactions (Sturtevant, 1994). However, there have been a few studies on the thermodynamic contribution of directly contacting residues to antigen-antibody interactions whose structure has been investigated in detail (Herron et al., 1986; Ito et al., 1993; Kelly and O'Connell, 1993; Tello et al., 1993; Tsumoto et al., 1994b). Furthermore, there is still no experimental approach for precise analysis of the thermodynamic role of Tyr residues in antibodies.

We have focused on the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, whose structural features have been analyzed by x-ray crystallography (Padlan et al., 1989) and have reported an expression system for the Fv fragment using a bacterial secretion system (Ueda et al., 1993; Tsumoto et al., 1994a). In the previous paper, we discussed the contribution of structurally perturbed antigenic residues upon antibody binding to the interaction using mutant antigens (Tsumoto et al., 1994b). We have described quantitatively the contribution of noncovalent bonds to the interaction using isothermal titration calorimetry and a feature that could not be explained by structural analysis alone.

In order to elucidate the mechanism of antigen-antibody interaction, it is important to study the role of the residues in the antibody that recognizes the antigen. X-ray crystallography has suggested that six Tyr residues localized in CDR of HyHEL10 participate in the recognition of HEL. In this study, we focused on four Tyr residues of the VH chain (HTyr-33, -50, -53, and -58) to study the role of Tyr residues. The Tyr residues at these sites have been suggested by structural analysis to contribute to the interaction in different ways (Fig. 1) (Padlan et al., 1989; Novotny, 1991). It is considered that thermodynamic study of the interaction using Fv fragments mutated at these sites will help to clarify the function of Tyr residues on the basis of structural analysis. The Tyr residues at positions 33, 50, 53, and 58 were systematically mutated with each of four amino acids: Trp, with a bulky volume and higher hydrophobicity; Phe, which lacks the hydroxyl group of Tyr; Leu, which has higher hydrophobicity and volume; and Ala, which has a smaller volume and hydrophobicity.


Figure 1: X-ray crystallographic analysis of the HyHEL10 Fv fragment-HEL complex. The drawing is from the Protein Data Bank (^3HFM), and the main chain of the Fv fragment-HEL complex is shown (Padlan et al., 1989). Blue and whiteribbon represent HEL and HyHEL10 Fv, respectively. The four Tyr residues studied here are drawn with side chain and numbered.



Here we report analysis of the interactions between antigen, HEL, and engineered HyHEL10 Fv fragments, using inhibition of the enzymatic activity of HEL and isothermal titration calorimetry. On the basis of thermodynamics, we describe how Tyr residues can show various and significant properties related to antigen binding with their aromatic rings and hydroxyl groups.


EXPERIMENTAL PROCEDURES

Materials

Enzymes for genetic engineering were obtained from Boehringer Mannheim, Takara Shuzo, or Toyobo. Helper phage M13KO7 for extracting single-stranded DNA was from Promega. Hen egg white lysozyme was from Seikagaku Kogyo Inc. (Tokyo). Micrococcus lysodekticus for measuring the enzymatic activity of lysozyme was obtained from Sigma. HEL-Sepharose for affinity chromatography was prepared from CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.). Oligonucleotide DNA primers were synthesized by a 381A DNA synthesizer (Applied Biosystems). A mutagenesis kit was obtained from Bio-Rad. All other chemicals used were of reagent grade appropriate for biochemical research.

Site-directed Mutagenesis of Tyr Residues Localized in CDRs of HyHEL10 VH

General recombinant DNA technology followed that of Sambrook et al.(1989) in principle. Site-directed mutagenesis was done according to the method of Kunkel(1985) using phagemid pTZ18U (Bio-Rad). Oligonucleotide DNA primers for mutagenesis are listed in Table 1. Correctness of mutations was confirmed by DNA sequencing using a Bca-BEST® sequencing kit (Takara).



Expression and Purification of Mutant Fv Fragments

For more convenient preparation, a modified expression procedure was used. An early stationary phase culture of Escherichia coli BL21(DE3) harboring pKTN2 (Tsumoto et al., 1994b) was centrifuged, resuspended in 2 YT medium (16 g of Bactotryptone, 10 g of yeast extract, 5 g of NaCl/liter of culture) with 100 mg/liter ampicillin and isopropyl-1-thio-beta-D-galactopyranoside at a final concentration of 1 mM, and then grown overnight at 28 °C (shaking for 18-20 h). Purification of mutant HyHEL10 Fv fragments from the culture medium was done as described previously (Tsumoto et al., 1994b). The procedures used for SDS-PAGE followed those of Laemmli(1970). Samples for SDS-PAGE were prepared as described previously (Tsumoto et al., 1994a).

Inhibition Assay of HEL Enzymatic Activity by Mutant Fv Fragments

The method used to measure the inhibitory activity of HyHEL10 mutant Fv toward HEL was that of Ueda et al.(1993).

Isothermal Titration Calorimetry

The thermodynamic parameters of the interactions between HEL and the mutant Fv fragments were determined by isothermal titration calorimetry using an OMEGA titration microcalorimeter (Wiseman et al., 1989) from MicroCal Inc. (Northampton, MA). The HEL at a concentration of approximately 5 µM in 50 mM phosphate buffer (pH 7.2) containing 0.2 M NaCl in a calorimeter cell was titrated with a 125 µM solution of the wild-type or mutant Fv fragments in the same buffer at five different temperatures (20, 25, 30, 35, and 40 °C). The ligand solution was injected 16 times in portions of 7 µl during 15 s. Thermogram data were analyzed by a computer program (Origin) supplied by MicroCal Inc. (Wiseman et al., 1989).

Estimation of Protein Concentration

The concentration of HEL was estimated using A = 26.5 (Imoto et al., 1972). The concentration of wild-type Fv fragment was estimated using A = 20.6 (Tsumoto et al., 1994b). The concentrations of Tyr-mutated Fv fragments were determined by the method of Lowry et al. (1951), using wild-type Fv fragment as a standard.


RESULTS

Expression and Purification of Mutant Fv Fragments

All mutants except HY50W and HY58W were obtained in the E. coli BL21(DE3) expression system. We attempted to express HY50W and HY58W mutants in E. coli several times under several conditions but failed to do so. Mutant HY33A and mutant HY50A could not be purified by affinity chromatography using HEL-Sepharose (Tsumoto et al., 1994b), although the expression levels of these mutants were very high (HY50A is shown in Fig. 2B), suggesting that these mutants are devoid of binding activity to HEL. All other mutants were expressed and purified according to the methods described previously (Tsumoto et al., 1994a, 1994b).


Figure 2: SDS-PAGE analyses of the mutated HyHEL10 Fv fragments purified. A, SDS-PAGE analyses of fractions obtained by affinity chromatography on HEL-Sepharose. The purification of the HY50F mutant HyHEL10 Fv fragment was shown. Concentrated culture supernatant (10 ml per 1 liter of culture) (lane2) was loaded onto 2.5 mL HEL-Sepharose (10 mg of HEL/ml of Sepharose 4B), which was previously equilibrated with 50 mM phosphate buffer (pH 7.2) containing 200 mM NaCl. The column was washed with a 20-fold of column volume the same buffer (lanes3 and 4), and with a 10-fold volume of 100 mM Tris-HCl (pH 8.5) containing 500 mM NaCl (lane5), and then the adsorbed protein was eluted with 100 mM glycine buffer (pH 2.0) (lanes6 and 7). Sample preparation for PAGE was done by the procedure of Tsumoto et al. (1994b) and then followed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue C-250. Lane1 is molecular size markers, and they are indicated in kDa. B, 15% SDS-PAGE analyses of expressed and purified mutant Fv fragments. Lanes1 and 4, culture supernatant of E. coli BL21 (DE3) expressed mutant Fv; lanes2 and 5, flow-through fractions of affinity chromatography; lanes3 and 6 eluted fractions. Lanes1-3 represent HY58F, and lanes4-6 represent HY50A. The molecular size markers are indicated in kDa.



SDS-PAGE analyses of the fractions of the affinity chromatogram on a HEL-Sepharose are shown in Fig. 2A. In the purification of the wild-type HyHEL10 Fv fragment by affinity chromatography using HEL-Sepharose, almost all Fv fragment expressed in E. coli was bound specifically to the HEL-Sepharose and was eluted (Tsumoto et al., 1994b). In fact, in the purification of the HY33F, HY53F, and HY58F mutant Fv fragments, all of the mutants expressed in E. coli were bound and eluted under the same conditions as that for the wild-type Fv (Fig. 2B, lanes1-3). In the case of HY53W, HY53L, HY33W, HY33L, HY50F, HY58L, and HY58A, a small amount of the protein flowed through HEL-Sepharose and leaked out during washing (HY50F is shown in Fig. 2B). In the case of purification of the HY53A and HY50L mutant Fv fragments, a significant amount of Fv fragments leaked out from the HEL-Sepharose, suggesting that these mutations reduced the association with the HEL. The HY33A and HY50A mutant Fv fragments were expressed, but flowed through the HEL-Sepharose (HY50A is shown in lanes4-6 of Fig. 2B). Thus, we could not use the HY50W, HY58W, HY33A, and HY50A mutant Fv fragments in this study. The yields of the mutant Fv fragments varied; that of HY53A or HY50L was 2-3 mg/1-liter culture, whereas that of HY58F or HY53F was more than 10 mg/1-liter culture.

Antigen Binding Activities of Mutated HyHEL10 Fv Fragments

The HyHEL10 Fv fragment has almost complete inhibitory activity toward its antigen, HEL, in a molar ratio of 1 Fv to 1 HEL, as described previously (Ueda et al., 1993; Tsumoto et al., 1994a, 1994b), resulting from the binding of Fv to the active site of HEL. Therefore, inhibition of the enzymatic activity of HEL by the mutant Fv fragments was investigated. The inhibition profiles for the mutant Fv fragments are shown in Fig. 3, and the results are summarized in Table 2.


Figure 3: Inhibition of HEL activity by VHTyr engineered HyHEL10 Fv fragments. HEL at 1.5 µM and the Fv fragments at various concentrations were incubated for 1 h, and mixed with a M. lysodekticus cell suspension. The process of lysis of M. lysodekticus cells by HEL was monitored by the decrease in absorbance at 540 nm at pH 7.2 and 25 °C. The remaining activity was measured and plotted against the molar ratio of Fv to HEL. Symbols are as follows: ‖‖‖, inhibition profile for the wild-type HyHEL10 Fv; , that for the HY58F; ▪, that for HY53L; , that for HY53A; ▴, that for HY58A; , that for HY53F.





Although the inhibitory activity of the HY58F mutant Fv fragment was slightly decreased in comparison with that of the wild-type Fv, the inhibitory activities of other mutants were significantly decreased. In particular, the inhibition ability of HY53A and HY50L was decreased by 3 orders of magnitude. Moreover, the extent of the inhibitory activities of the mutants at the same position (i.e. each site of 33, 50, 53, and 58 of VH chain) differed according to the residues substituted (Table 2). Although the inhibition level of the mutant Fv fragments substituted with Trp, Phe, or Leu at position 53 was almost identical and slightly lower than that of the wild-type Fv, substitution with Ala almost totally abolished the inhibitory activity. This suggests that the hydrophobicity of the side chain at site 53 of VH is crucial for binding. On the other hand, the inhibition level of the Phe mutant at position 33 was slightly decreased, and substitution with Trp or Leu markedly decreased the inhibition, suggesting that complementary association of an aromatic ring at site 33 of VH in HyHEL10 is significant for recognition of HEL.

These results indicate that each mutation weakened the interaction with HEL and that the lower yields of mutant Fv fragments obtained by purification using the affinity chromatography were in principle consistent with the results of the inhibition assay. The lowered inhibitory activities of the mutants indicate that the Tyr residue at each of the four positions plays a key role in the HyHEL10-HEL interaction, although the extent of inhibition differs at each site.

Thermodynamic Analyses of the Interactions between Tyr-mutated Fv Fragments and HEL

The magnitudes of inhibition of HEL activity increased with increasing molar ratio of Fv to HEL in all mutant Fv fragments, even HY50L and HY53A (Table 2), suggesting that the degree of inhibition reflects the strength of binding of Fv with HEL. In order to estimate quantitatively the interaction between Fv and HEL and to obtain the thermodynamic parameters for the interaction, we performed calorimetric titration for the binding of Fv and HEL at a constant temperature using an OMEGA calorimeter. Calorimetric titration was performed at five different temperatures. Three typical titration curves are shown in Fig. 4. The binding constant and the enthalpy change upon antigen-antibody binding can be obtained according to the method of Wiseman et al.(1989). We can obtain the heat capacity change (DeltaCp) of the binding from the temperature dependence of the enthalpy change (DeltaH) (Fig. 5). Thermodynamic parameters at 30 °C calculated from the titration curves are represented in Table 3.


Figure 4: Typical titration curves of the interactions between VH Tyr engineered HyHEL10 Fv fragments and HEL at 30 °C, pH 7.2. Calorimetric titration was performed under the condition described under ``Experimental Procedures.'' The enthalpies (heating value of binding) produced by injection of Fv to HEL were plotted against the injection number. A, the titration curve of HY53L; B, that of HY33W; C, that of HY53A.




Figure 5: Temperature dependence of enthalpy change of the interactions between HTyr mutant HyHEL10 Fv fragments and HEL at pH 7.2. The values of negative enthalpy were plotted against temperature. A-D represent the plots of HTyr53, HTyr33, HTyr50, and HTyr58, respectively. The slopes were obtained by linear regression. Symbols for each mutation are as follows: ▪, enthalpy for the wild-type Fv; , that for the Trp mutant; bullet, that for the Phe mutant; , that for the Leu mutant; ▴, that for the Ala mutant.





The stoichiometry (n) of the interaction between each mutant Fv and HEL was nearly equal to unity. However, the association constants (K) of all the mutant Fv fragments were lower than those of the wild-type Fv and also differed from each other according to the residues substituted. The extent of the decrease in the K values for the mutant Fv fragments corresponded to those of their inhibitory activities (Table 2). This indicates that the lowered inhibition levels result from the lowered binding constant.

HTyr-53

For the Phe and Leu mutants, the binding constant (K) and Gibbs energy change (DeltaG = -RTlnK), which are quantitative measures of the strength of binding with HEL, were slightly smaller than those of the wild-type Fv and similar to each other in the two Fv fragments. The slight decrease of K originated from the decrease in the negative enthalpies (DeltaG = DeltaH - TDeltaS), since the decreases of negative DeltaH (7.1-8.6 kJ mol) exceeded the decreases of negative TDeltaS (4.1-5.9 kJ mol). The negative DeltaCp value of the Phe mutant Fv (-0.98 kJ molK) was smaller than that of the wild-type, whereas that of the Leu mutant Fv (-1.43 kJ molK) was almost the same as that of the wild-type. Although K and DeltaG of the Trp mutant were almost identical to those of the Phe and Leu mutants, the changes in DeltaH and DeltaS were different from those of all the mutant Fv fragments examined; the negative values of DeltaH and TDeltaS were higher than those of the wild-type. This suggests that introduction of the bulky and strong hydrophobic indole ring at this position affects the binding with HEL in a different manner. The Ala mutant Fv showed markedly weakened interaction with HEL. The binding constant was 2 orders of magnitude lower than that of the wild-type, which was due mainly to a remarkable decrease in DeltaH. This indicates that the specific interactions by hydrophobic side chain at site 53 are significant for the interaction with HEL.

HTyr-33

The K values of the three mutant Fv fragments substituted with Trp, Phe, or Leu were markedly decreased by 1 order of magnitude compared with the wild-type Fv. The DeltaH values of the three mutants were similar to each other and reduced their negative values (DeltaDeltaH was over 18 kJ mol at 30 °C (Table 3). This enthalpic disadvantage was partially compensated by the resulting reduction of entropic loss. The negative DeltaCp values of the three mutant Fv fragments were similar to each other and higher than that of the wild-type. It was found that the three mutant Fv fragments had markedly lowered strength of association with HEL at a similar level. These results suggest that the hydroxyl group at position 33 of VH is crucial for the HyHEL10-HEL interaction.

HTyr-50

The negative enthalpy of the interaction between the HY50F mutant Fv and HEL was about 32 kJ mol smaller than that of wild-type Fv (Table 3), whereas the interaction between the HY50L mutant Fv and HEL was about 38 kJ mol smaller. The entropic losses were similar to each other (TDeltaDeltaS was about 25 kJ mol) and smallest among the mutants examined. The large decrease of negative DeltaH resulted in a marked decrease of K; 1 order of magnitude lower in the Phe mutant and 2 orders lower in the Leu mutant. Thus, the hydroxyl group of HTyr-50 was considered to be significant for the interaction.

HTyr-58

The thermodynamic parameters of the interaction between HY58F and HEL were almost indistinguishable from those of the wild-type Fv (Table 3). On the other hand, the binding enthalpy of the interaction between HY58L or HY58A Fv fragments and HEL was about 29 or 19 kJ mol, respectively, smaller than that of the wild-type, resulting in a 1-order decrease of affinity. This suggests the significance of the aromatic ring at site 58. The negative DeltaCp values of the Leu and Ala mutants were about 0.40 kJ mol smaller than that of the wild-type.


DISCUSSION

Contribution of Direct Contact of the Tyr Residue at Each Site to the Interaction Is in Principle Enthalpy-gaining

The enthalpy changes (DeltaH) shown in Table 3were plotted against the entropy changes (TDeltaS) (Fig. 6, A-D). Each plot showed good linearity except that for HY53W, and the slopes were estimated to be 1.80 (HTyr-53), 1.45 (HTyr-33), 1.39 (HTyr-50) and 1.32 (HTyr-58). These slopes were larger than 1 (complete compensation), suggesting that the negative enthalpic gain by the Tyr residues dominates their contributions to the interaction. As discussed by Herron et al.(1986), Kuroki et al.(1992), and Brummell et al.(1993), changes in enthalpy and entropy tend to compensate each other to some degree. Namely, the enthalpic advantage obtained by forming hydrogen bonds and van der Waals' interactions is compensated by the entropic loss in fixing the vibrational and translational freedom upon binding. However, these slopes suggest that the Tyr residues, which directly participate in recognition of the antigen, make positive contributions to the interaction by gaining more enthalpic energy at the cost of entropic loss.


Figure 6: Enthalpy-entropy compensation plots for the interactions between VH Tyr engineered HyHEL10 Fv fragments and HEL at 30 °C, pH 7.2. -DeltaH values in Table 2were plotted against -TDeltaS. The slope was obtained by linear regression. A-D are the plots for HTyr53, HTyr33, HTyr50, and HTyr58, respectively. The slope (X) and the intercept (Y) for each plot are as follows: (X, Y) = (1.80, 17.6) (HTyr53), (1.45, 31.8) (HTyr33), (1.39, 34.1) (HTyr50), and (1.32, 36.3) (HTyr58).



Tyr Residues May Contribute to the Coupling of Local Folding to Site-specific Binding

In the previous paper, it has been reported, by using the mutants substituted Trp-62 and Asp-101 with Gly, that the formation of the HyHEL10 Fv fragment-HEL complex couples the local conformational changes of the proteins (Tsumoto et al., 1994b). Since the structure of the HyHEL10 Fv fragment has not yet been solved, three possibilities can be considered. The first is perturbation of the antigenic structure upon binding. Structural analysis has indicated that HTyr-53 contributes to the rotation of the indole ring of Trp-62 of HEL upon binding. The second is local conformational change of antibody for recognition of antigen upon binding (i.e. induced fit of antibody) (Rini et al., 1992), and the third is structural perturbation of both antigen and antibody upon binding. To examine local folding coupled with the site-specific binding of mutant Fv fragments to HEL, we analyzed the data obtained from titration calorimetry according to the method of Spolar and Record(1994). The results are summarized in Table 4. In all interactions between mutant Fv and HEL, the numbers of residues involved in the coupling folding were decreased, suggesting that the Tyr residue at each site participate in the coupling folding. In the HyHEL10-HEL interaction, no major conformational change of antigen, HEL has been observed (Padlan et al., 1989). Then, Tyr residues at each site may actively cause function to local conformational changes of antibody.



The decreasing degrees differed among the mutants. For instance, three mutants at site 33 had almost the same R value, suggesting that the hydroxyl group contributed to the local folding. Although this also applied to the case of HTyr-50 mutants, the R value was somewhat decreased in comparison with that of the wild-type, suggesting the significant role of the hydroxyl group at site 50 of VH in the local folding upon binding. On the other hand, in the case of HTyr-58 mutants, the R value of Phe mutants showed only a small difference from that of the wild-type. However, the R values of the Leu and Ala mutants were significantly different from that of the wild-type, suggesting the significance of the aromatic ring at site 58 for the interaction.

Role of the Hydroxyl Group of Tyr Residues at Positions 33 and 50 in Antigen Binding: Study Using Phe Mutants

At positions 33 and 50, the binding constants of the interactions between Phe mutants and HEL were observed to be 1 order of magnitude lower (Table 3). In the case of HY33F Fv, the negative enthalpy was about 18 kJ mol smaller than that of the wild-type. Enthalpic change due to the formation of hydrogen bonds has been estimated to be about -17.6 kJ mol by site-directed mutagenesis and titration calorimetry (Connelly et al., 1994). The hydroxyl of HTyr-33 has been proposed to form a hydrogen bond with the main chain of Lys-97 in HEL (Padlan et al., 1989). Thus it can be considered that the observed enthalpic loss for the HY33F Fv results from the disappearance of the hydrogen bond.

In the case of HY50F, the decreases of -DeltaH and -TDeltaS were larger than those for HY33F. Structural analysis has suggested that the hydroxyl of HTyr-50 forms two hydrogen bonds with the side chain of Arg-21 and the main chain of Ser-100 in HEL (Padlan et al., 1989). Therefore, failure to form these two hydrogen bonds may decrease the -DeltaH and -TDeltaS of the interaction to a much greater extent than the case of HTyr-33.

In addition, analyses using the methods of Spolar and Record also supported the significance of the hydroxyl at these sites in the local folding induced by the interaction (Table 4). From these results, the hydroxyl groups of Tyr at positions 33 and 50 are concluded to play a critical role in the interaction by forming hydrogen bonds with the antigen, HEL.

Contribution of the Aromatic Ring of Tyr Residues to the Association: Data from Leu Mutants

At positions 33 and 50, the enthalpic changes in the interactions of the Leu mutants were about 6 kJ mol smaller than that of Phe mutants, and the entropic changes of the Leu mutants were almost the same as those of the Phe mutants (Table 3), suggesting that the aromatic ring of Tyr at these sites contributes to the Gibbs energy by gaining enthalpic energy and decreasing the entropic loss. The inhibition level of the Leu mutants at these sites was 1 order of magnitude lower than that of the Phe mutants (Table 2), resulting from the aromatic ring removal and introducing an aliphatic side chain at these sites. It might also be assumed that a slight conformational change is introduced by substituting Tyr with Leu, and that this affects the covering of the substrate binding cleft of HEL.

On the other hand, the binding constant of the interaction between the HY58L mutant Fv and HEL was 1 order of magnitude lower than that of the wild-type, although no significant effect was observed in the HY58F mutant Fv, suggesting that the aromatic ring is crucial at position 58. Structural analyses have suggested that an amino-aromatic interaction (Burley and Petsko, 1988) between -electrons of the aromatic ring and the amino group of Arg-21 is formed (Padlan et al., 1989). In addition, an aromatic ring cluster is reported to be formed in the contact region around HTyr-58, suggesting that the aromatic ring may stabilize the local CDR structure through aromatic-aromatic interaction (Burley and Petsko, 1988). The analyses of Spolar and Record also supported a critical role of the aromatic ring of the Tyr residue at site 58 in the coupling folding (Table 4).

Effect the Hydrophobicity of Tyr Residues on the Interaction: Data from Ala Mutants

At all four sites, substitutions of Tyr residues with Ala weakened the interaction greatly. At sites 33 and 50, the Ala mutant could not be purified by affinity chromatography, indicating that these mutations resulted in loss of binding activity to HEL. Both the volume and hydrophobicity of the Ala residue are smaller than those of Trp, Tyr, Phe, and Leu. In the case of site 58, the inhibition levels and binding constants of the Ala and Leu mutants were 1 order of magnitude lower than those of wild-type Fv ( Table 2and Table 3), indicating the significance of the aromatic ring.

In the case of site 53 of VH, Phe and Trp (as described below) can play the role of Tyr, and even Leu function in this role. However, Ala cannot perform this role. The side chain volume of Ala is about half of that of Tyr, and the hydrophobicity of Ala residue is one-fourth of that for Tyr residue (Nozaki and Tanford, 1971). Therefore, it can be concluded that the larger volume or hydrophobicity, or both of them at the site 53, are crucial for the interaction.

Effect of Introducing Bulky Side Chain into the Tyr Site: Data from Trp Mutants

In the case of site 53 of VH, both negative enthalpy and entropy were increased by substitution with Trp, suggesting that the location of a more bulky side chain at site 53 of VH might result in the formation of tighter bonds at the binding site. In this case, the greater entropic decrease overcame the enthalpic decrease, resulting in a lower binding constant. However, at site 33 of VH, a reduced enthalpic change and Gibbs energy were observed, suggesting that the size of the aromatic ring of Tyr is best fitted at site 33 of VH. Substituting Tyr with Trp at sites 50 and 58 resulted in inability of expression in the E. coli system. Since structural analysis has shown that favorable aromatic-aromatic interaction seems to be formed at these sites (Padlan et al., 1989), structural perturbation induced by the substitutions with Trp at these sites might decrease the stability of the mutant Fv fragments, resulting in inability to express in E. coli.

Tyr Residue at Each Site Makes a Significant Contribution to the Interaction

From the results of affinity chromatography, inhibition assay of HEL activity, and titration calorimetry of the Tyr mutants at the four sites, the various and significant roles of Tyr localized in CDR in the interaction with HEL can be summarized as follows. 1) Sites 33 and 50, the hydroxyl group of Tyr stabilizes the binding by forming hydrogen bonds with HEL. The aromatic ring also contributes to the Gibbs energy with a gain of enthalpic energy. 2) Site 53, since Trp, Phe, and Leu can take the place of Tyr but Ala cannot, the hydrophobic interaction by the side chain may be significant. 3) Site 58, the hydroxyl group seems not to be significant, whereas the aromatic ring is significant, perhaps because of the amino-aromatic interaction with Arg-21 of HEL and stabilization of local CDR structure by aromatic-aromatic interaction.

In conclusion, it was revealed here that the Tyr residues localized in CDRs of HyHEL10 VH play various significant roles in the association with the antigen, which are dependent on the structural features of the antigenic residues: formation of a hydrogen bond by the hydroxyl group, hydrophobic interaction due to its higher hydrophobicity, van der Waals' interaction due to its larger volume, and stabilization of local structure due to its aromatic ring.

In ligand-antibody interactions, it has been suggested that Tyr residues unusually distributed in the contact region make favorable aromatic-aromatic interaction (Burley and Petsko, 1988), van der Waals' interactions with ligands and hydrogen bonds (Rini et al., 1992; Satow et al., 1986; Herron et al., 1989) and form hydrophobic environment with indole rings of Trp (Herron et al., 1989). Moreover, recent x-ray crystallographic studies of protenacious antigen-antibody interactions have proposed that a lot of Tyr residues in CDRs make van der Waals' interactions by the aromatic ring and hydrogen bonds by the hydroxyl group (Chitarra et al., 1993; Bhat et al., 1994). Then, site-directed mutagenesis and thermodynamic study of these interactions might reveal that some of the various roles of Tyr residues of HyHEL10 reported here are generalized to a lot of antigen-antibody interactions.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-77, Japan.

^1
The abbreviations used are: CDR, complementarity determining region; Fv, fragment of variable region of immunoglobulin; VH, fragment of variable region of heavy chain; VL, fragment of variable region of light chain; HEL, hen egg white lysozyme; HY53W, mutant Fv fragment in which Tyr of VH chain is substituted with Trp at position 53; PAGE, polyacrylamide gel electrophoresis.


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