(Received for publication, May 16, 1995; and in revised form, July 12, 1995)
From the
To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45 °C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.
In affinity maturation of the immune response, the average
affinity of the immunized serum generally increases with time after
immunization(1) . To investigate the relationship between the
primary sequence diversity of antibodies and the progressive change in
binding affinity, extensive analyses of antibodies have been carried
out by using several haptens, 4-hydroxy-3-nitrophenylacetyl
(NP)()(2, 3, 4, 5) , p-azophenylarsonate(6) ,
phosphorylcholine(7) , and 2-phenyl-5-oxazolone(8) .
A series of anti-NP mouse monoclonal IgG antibodies used in the
present study were produced by the immune response of C57BL/6 mice
against NP coupled to T cell-dependent carrier, chicken -globulin (9, 10) . The variable regions of the primary response
anti-NP antibodies show low affinity for NP and carry few, if any,
somatic mutations(2, 4) , whereas those of the
secondary response anti-NP antibodies usually exhibit increased
affinity for NP and are somatically mutated(5) . The secondary
response antibodies are divided into two groups by carrying or lacking
a somatic Trp
Leu exchange at position 33 in the variable region
of the heavy chain(11, 12) . In the present study we
compare N1G9, a primary response anti-NP antibody, with 3B44 and 3B62,
which are secondary response anti-NP antibodies with and without Trp
Leu exchange, respectively.
Thermodynamic aspect of
antigen-antibody association is essential in order to understand the
mechanism of the high affinity and specificity of antigen-antibody
interaction. Thermodynamic parameters such as Gibbs free energy change,
G, enthalpy change,
H, entropy change,
S, and heat capacity change,
Cp, can
provide useful information to identify fundamental forces involved in
the antigen-antibody interaction. For instance, the magnitude of
Cp is usually related to the contribution of the
hydrophobic effect to molecular association (13, 14, 15, 16) .
With the recent improvement in the sensitivity and reliability of the calorimeter(17, 18) , isothermal titration calorimetry (ITC) has become a powerful tool for the direct measurement of thermodynamic parameters in various biological interactions, such as protein-protein interactions(19, 20, 21, 22) , oligosaccharide-lectin associations(23, 24, 25) , and ligand binding to proteins(26, 27) . Recently this method has been applied to the quantitative thermodynamic analyses of antibody binding to various types of antigens, e.g. haptens(28, 29, 30, 31) , oligosaccharides(32, 33, 34) , and proteins(35, 36, 37, 38, 39, 40, 41, 42, 43, 44) . However, no calorimetric studies have been reported on the antigen-antibody interaction in affinity maturation.
In the present
study we show the ITC analyses of the antigen-antibody associations in
affinity maturation. We examined the interactions between
4-hydroxy-3-nitrophenylacetyl caproic acid (NP-Cap) antigen and the Fab
fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62. On the
basis of the obtained thermodynamic data, we have found that the
binding constants, K, for NP-Cap
correlate with each of the
H,
Cp, and
Su values. As the logarithm of the K
values increases in the course of affinity maturation, the
magnitudes of the corresponding
H,
Cp, and
Su values increase almost linearly. Although the
interactions between a series of monoclonal antibodies and their same
antigen have been investigated in several
cases(35, 36, 37, 39, 43, 44, 45) ,
the linear relationship between log K
and
each of
H,
Cp, and
Su shown
in the present study has not yet been observed. On the basis of this
correlation of the thermodynamic data along with the previously
reported nuclear magnetic resonance (NMR) data(46) , we will
discuss the mechanism of the affinity maturation.
Figure 1: Typical isothermal titration calorimetric profiles of the interaction between NP-Cap and Fab(3B62) at 30.0 °C. a, the NP-Cap solution (1.0 mM in 5 mM sodium phosphate buffer, 200 mM sodium chloride, pH 8.0) was injected 20 times in 5-µl increments into 38 µM Fab(3B62) solution, which was dialyzed against the same buffer. Injections were occurred over 10 s at 3-min intervals. b, integrated areas for the above peaks were plotted against the molar ratio (NP-Cap)/(Fab(3B62)). The data were fitted using a nonlinear least-squares method.
The magnitudes of the
S and
G values are dependent on the
concentration units for the standard state. In order to obtain unitary
entropy change,
Su, and unitary Gibbs free energy change,
Gu, which are independent of the concentration units
chosen for the standard state since, in essence, solute concentrations
are measured in mole fraction units, we used the following
equations(48) :
The cratic contribution to the entropy change, R lnX, is R ln(1/55.6) =
-7.98 cal mol
K
, where 55.6 M is the concentration of water in dilute aqueous
solution(13) . In the case of Fig. 1
Su and
Gu are -27.5 ± 0.5 cal
mol
K
and -12.0 ±
0.1 kcal mol
, respectively. The thermodynamic
parameters for the interaction between NP-Cap and each of Fab(N1G9) and
Fab(3B44) were obtained in the same way.
Figure 2:
Thermodynamic parameters (H (
),
Gu (
), and
-T
Su (
)) for the associations
between NP-Cap and each of Fab(N1G9), Fab(3B44), and Fab(3B62) as a
function of temperature. All experiments were performed in 5 mM sodium phosphate buffer, 200 mM sodium chloride, pH 8.0,
at the experimental temperature. The solid lines were obtained
from a linear least-squares regression.
Cp was calculated
from the slope of the regression line of
H (
).
The
associations between NP-Cap and the three anti-NP Fab are mainly driven
by favorable negative changes in H. The negative
H values decrease with increasing temperature and show
linear dependence on temperature in the range of 25-45 °C (Fig. 2). The
Cp value for each interaction can be
determined from the slope of the temperature dependence of
H. The negative
Cp values of -309
± 27, -363 ± 12, and -415 ± 12 cal
mol
K
are observed for Fab(N1G9),
Fab(3B44), and Fab(3B62), respectively (Table 1).
Fig. 3shows that the K values for NP-Cap
correlate with each of the
H,
Cp, and
Su values. As the logarithm of the K
values increases in the order of Fab(N1G9), Fab(3B44), and
Fab(3B62), the magnitudes of the corresponding
H,
Cp, and
Su values increase almost linearly.
Figure 3:
Correlations between the log K values at 25 °C and each
absolute value of
H at 25 °C (a),
Cp (b), and
Su at 25 °C (c).
Figure 4:
Dependence of the log K values of Fab(N1G9) (
),
Fab(3B44) (
), and Fab(3B62) (
) for NP-Cap upon the
logarithm of the sodium chloride concentration at 30 °C. All
experiments were performed in 5 mM sodium phosphate buffer, pH
8.0, containing the experimental concentration of sodium
chloride.
In the present study we have carried out the ITC analyses of
the antigen-antibody associations in affinity maturation, in order to
understand the mechanism of affinity maturation. The present results
reveal that all of the associations between NP-Cap and the three Fab
are mainly driven by favorable negative changes in H. Van
der Waals interactions and hydrogen bondings are usually considered to
be the major potential sources of the negative
H values (40, 43, 50) . Thus, we suggest that van der
Waals interactions and hydrogen bondings play a fundamental role in the
interactions between NP-Cap and the three Fab. Also, the increase in
the magnitude of the negative
H with the increase in log K
(Fig. 3a) suggests that, in the
course of the affinity maturation, the increase in the van der Waals
interactions and hydrogen bondings promotes the increase in the binding
affinity of the anti-NP antibody.
The negative Cp values in Table 1are within the range of -100 to
-650 cal mol
K
, which were
previously reported for various antigen-antibody associations (28, 29,
35, 37, 38, 40, 41, 43-45). In general, the negative
Cp values for protein folding and protein-ligand association are
proportional to the reduction in water-accessible nonpolar surface
areas of the molecules, and related to the contribution of hydrophobic
effect to molecular
association(13, 14, 15, 16, 51) .
In order to interpret our data quantitatively, the empirical method of
Sturtevant (52) was used to estimate the hydrophobic and
intramolecular vibrational contribution to
Cp (Table 2). For all the three Fab, the calculated hydrophobic
contribution to
Cp is larger than the calculated
vibrational contribution. Therefore, we suggest that the observed
negative change in
Cp may primarily result from the
hydrophobic effect, that is, the decrease in solvent exposure of both
the aromatic antigen and the nonpolar groups in the binding site of the
three Fab caused by the antigen-antibody association. Furthermore, the
increase in the magnitude of the negative
Cp with the
increase in log K
(Fig. 3b)
suggests that, in the course of the affinity maturation, the increase
in the hydrophobic effect contributes to the increase in the binding
affinity of the anti-NP antibody. This is consistent with the previous
NMR result that the binding site of NP-Cap is located in a similar
position, but the combining site of Fab(3B62) with higher affinity for
NP-Cap is composed of more Tyr residues than that of Fab(N1G9) with
lower affinity for NP-Cap(46) .
The hydrophobic effect,
which drives the association of nonpolar surfaces of molecules by
excluding water from the interface, would contribute to the positive
change in Su. However, we observed the unfavorable
negative
Su values in the range of 25-45 °C, as
shown in Table 1. Negative
Su has been observed
previously for other antigen-antibody interactions (29, 30, 31, 32, 35, 36, 39, 40, 41, 42, 43, 44) .
Consequently, we conclude that some other factors should counteract the
hydrophobic effect and make larger contributions to the negative
Su. Such effect to the negative
Su can be
produced by the following factors: 1) the constraint of intramolecular
vibrational flexibility of Fab due to the antigen binding(52) ;
2) the reduction in the translational and overall rotational degrees of
freedom upon the complex formation(53, 54) ; and 3)
the conformational freezing of the amino acid residues of Fab caused by
the antigen binding(55) . The previously reported estimate of
the factor 2) was almost constant for different antigen-antibody
complexes (T
S
= 7-11
kcal mol
, where
S
is an
amount of translational and overall rotational entropy change) (53, 54, 55) . The estimation of the factor 3
was previously reported in the interactions between lysozyme and a few
anti-lysozyme monoclonal antibodies(55) . We applied the
empirical method of Sturtevant (52) in order to estimate the
hydrophobic and intramolecular vibrational (described above as the
factor 1) contribution to
Su (Table 2). For all
three Fab, the sign of the calculated hydrophobic contribution is
positive, but that of the calculated vibrational contribution is indeed
negative.
The obtained K values shown in Table 1increase in the order of Fab(N1G9), Fab(3B44), and
Fab(3B62), which is consistent with the previously reported
results(2, 4, 5) . As the logarithm of the
sodium chloride concentration increases, the logarithm of the K
values of the three Fab for NP-Cap decrease
linearly. The dependence of the K
values on the
sodium chloride concentration is similar for the three Fab (Fig. 4). These results suggest that the electrostatic effect is
involved in the antigen-antibody associations, but the proportion of
the electrostatic effect to the NP-Cap binding is similar for the three
Fab. We conclude that, in the course of the affinity maturation, the
electrostatic effect does not significantly contribute to the increase
in the binding affinity of the anti-NP antibody.
We have found a
linear correlation between log K and each of
H,
Cp, and
Su, as shown in Fig. 3. As the logarithm of the K
values
increases in the course of affinity maturation, the magnitudes of the
corresponding
H,
Cp, and
Su values increase almost linearly. Although the interactions between
a series of monoclonal antibodies and their same antigen have been
investigated in several
cases(35, 36, 37, 39, 43, 44, 45) ,
the linear relationship between log K
and each of
H,
Cp, and
Su shown in the
present study has not been observed yet.
This linear relation of log K,
H, and
Cp (Fig. 3, a and b) implies that the
surface complementarity of the anti-NP antibody with the antigen
increases in the course of the affinity maturation, which may reflect
increased van der Waals interactions and hydrogen bondings between
specific functional groups, and increased hydrophobic interactions with
more exclusion of water molecules from the interface. However, the
constancy of the electrostatic effect in the course of the affinity
maturation (Fig. 4) suggests that the number of electrostatic
interactions involved in the interface remains unchanged with the
increase in the surface complementarity. On the other hand, we observed
more pronounced effect of unfavorable negative
Su in the
course of the affinity maturation (Fig. 3c). This
apparently contradicting effect seems feasible, because more enhanced
surface complementarity would make more restraint in the intramolecular
vibrations of the complex.
We conclude that, in the course of the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity. The attainment of a better surface complementarity may be produced by an increase in the number of Tyr side chains in the antibody combining site.