(Received for publication, February 13, 1995; and in revised form, May 16, 1995)
From the
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.
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) ( 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 (
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.
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.
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;
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.
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;
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.
Figure 6:
Enthalpy-entropy compensation plots for
the interactions between VH Tyr engineered HyHEL10 Fv fragments and HEL
at 30 °C, pH 7.2. -
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.
In the case of HY50F, the decreases of - 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.
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
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.
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.
)(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.
HFM), 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.
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-
-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.
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).
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.
, that for the HY58F; ▪, that for
HY53L;
, that for HY53A; ▴, that for HY58A;
, that for
HY53F.
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 (Cp) of the binding
from the temperature dependence of the enthalpy change (
H) (Fig. 5). Thermodynamic parameters at 30
°C calculated from the titration curves are represented in Table 3.
, that for the Trp mutant;
, that for the Phe mutant;
, that for the Leu mutant;
▴, that for the Ala mutant.
HTyr-53
For the Phe and Leu mutants, the binding
constant (K) and Gibbs energy change (G =
-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 (
G =
H - T
S),
since the decreases of negative
H (7.1-8.6 kJ
mol
) exceeded the decreases of negative T
S (4.1-5.9 kJ mol
). The negative
Cp value of the Phe mutant Fv (-0.98 kJ
mol
K
) was smaller than
that of the wild-type, whereas that of the Leu mutant Fv (-1.43
kJ mol
K
) was almost the
same as that of the wild-type. Although K and
G of the Trp mutant were almost identical to those of the Phe and
Leu mutants, the changes in
H and
S were
different from those of all the mutant Fv fragments examined; the
negative values of
H and T
S 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
H. 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 H values of the three mutants were similar to each other and reduced
their negative values (
H 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
Cp 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 (T
S was about 25 kJ
mol
) and smallest among the mutants examined. The
large decrease of negative
H 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
Cp values of the Leu and Ala mutants
were about 0.40 kJ mol
smaller than that of the
wild-type.
Contribution of Direct Contact of the Tyr Residue at
Each Site to the Interaction Is in Principle
Enthalpy-gaining
The enthalpy changes (H) shown in Table 3were plotted against the entropy changes (T
S) (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.
H values in Table 2were plotted against -T
S. 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.
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.
H and -T
S 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 -
H and -T
S of the interaction to a much greater
extent than the case of HTyr-33.
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.
-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.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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.