(Received for publication, November 27, 1995; and in revised form, December 18, 1995)
From the
In a comparative study, the thermodynamic parameter,
V, was obtained using hydrostatic pressure-induced
dissociation of fluorescein (Fl) from the active site of monoclonal
antibody (mAb) 9-40 and its mutant and native derivatives equilibrated
at six pH values (8.0, 7.5, 7.0, 6.5, 6.0, and 5.5) and four
temperatures (35, 25, 15, and 5 °C). mAb 9-40 and its Fab and
single-chain Fv (scFv) derivatives at pH 8.0 were found to have
identical Fl dissociation behavior under pressure as a function of
temperature. The pressure dissociation at 25 °C as a function of pH
showed a sigmoidal dependence of
V with a midpoint value
at pH 7.4 for mAb 9-40. Comparison of experimental results for scFv
9-40/212 with its mutant scFv 9-40/212
indicated that
the pH dependence of mAb 9-40 was due to the titration of His-34L in
the active site. Iodide quenching of bound Fl showed that the hapten in
this active site was solvent accessible. Imperfect packing, which leads
to increased conformational dynamics, was determined as a possible
cause of the low affinity for mAb 9-40.
The understanding of antibody (Ab) ()structure-function has been enhanced during the past two
decades through the advent of hybridoma
methodology(1, 2) , the increase in resolved atomic
structures due primarily to x-ray crystallography (3, 4, 5, 6) , generation of Ab
structural derivatives such as single-chain Fv (scFv)
fragments(7, 8) , and site-specific mutagenesis of
well characterized Abs(9, 10) . However, despite these
significant advances, a comprehensive understanding of all the factors
that directly impact the binding of antigen remain ill defined. Such
factors as domain-domain interactions, binding affinity (K
) as it correlates with primary and
secondary interactions(11) , the total effect of somatic
mutations, and the structure-function role of the first constant
domains in both the heavy and light chains remain unresolved. The role
and magnitude of these parameters have been inferred from a spectrum of
studies, but direct measurements have not been made that correlate the
results with variable domain or protein dynamics(12) .
To
experimentally isolate these parameters for comparative and direct
quantitative measurements, model Ab reagents must be carefully selected
so that the results can be integrated into meaningful principles. The
conclusions from this study were based on two anti-fluorescein
(anti-Fl) mAbs, mAb 4-4-20 and mAb
9-40(13, 14, 15) , which are highly related
at the idiotypic or unliganded level, reflecting nearly identical
primary structures in both the heavy and light chains(14) .
However, these Abs were only marginally related at the metatype or
liganded level, suggesting significantly different conformations of the
liganded state(16, 17) . Primary structural studies
have also shown that 4-4-20 is a high affinity somatic mutant of the
lower affinity germ line 9-40 molecule(15) . The affinities of
these two Abs differ by 1000-fold for the Fl ligand with the value of
2.8 10
M
(10) and 2
10
M
(14) for 9-40 and 4-4-20,
respectively.
Comparatively, in mAb 9-40 the heavy chain variable (V) region of the amino acid sequence differs by
nine amino acid residues from 4-4-20 with most of the differences
localized in the HCDR3 hypervariable region, whereas the variable light
chain (V
) differed by only two amino acid
residues(14, 15) . Based on the crystal structure of
4-4-20, it was shown that only one 4-4-20 contact residue (Arg-34L) was
not present in 9-40 (His-34L). In summary, 9-40 provided an opportunity
to determine a set of rules that define affinity maturation by making a
comparison of a low affinity Ab to a high affinity Ab that were
structurally and sequentially similar.
In an effort to help
establish a set of rules specifying structure-function relationships
that may be eventually used for Ab engineering, hydrostatic
pressure-dependent behavior of mAb 9-40 and its structural derivative
scFv 9-40, a genetically engineered derivative of the anti-Fl mAb 9-40 (9, 10) comprised of the V and V
domains of the corresponding mAb joined by a
short polypeptide linker (7, 8) was studied. From
crystallographic studies, it was known that when Fl was bound, Arg-34L
in 4-4-20 was salt linked to the enolic
oxygen(18, 19) . The crystal structure of 9-40 was not
yet available, and therefore the role of His at the 34L position in
connection to Fl was not known. However, the influence of the contact
residue His-34L was monitored by comparing scFv9-40/212 with
scFv9-40/212
. Specifically, hydrostatic pressure was
used to induce dissociation of Fl as a function of pH from the active
site of mAb 9-40 and its native and mutant derivatives, scFv9-40/212
and scFv9-40/212
, respectively. The thermodynamic
parameter, change in partial molar volume (
V), was found
to be pH-dependent for mAb 9-40.
Because the affinities of mAb 9-40 and its derivatives, Fab 9-40 and scFv 9-40/212, for Fl were identical(10) , the influence of IgG constant domains on Ab stability were also investigated. Hydrostatic pressure was used to study the effect of IgG constant domains on 9-40 binding without the additional complication of affinity as a variable. In this study, pressure induced dissociation behavior as a function of temperature of mAb and its Fab and scFv derivatives were identical, within error. Finally, compared with 4-4-20, iodide quenching of bound Fl in 9-40 and 4-4-20 also showed Fl was more solvent-accessible in 9-40.
where I was the intensity at zero quencher
concentration, I was the intensity at quencher concentration
[Q], k
was the bimolecular
constant, and
was the lifetime of the fluorophore.
where R is the ratio of the fluorescence quantum yields
of free and bound forms, r is the anisotropy at each pressure,
and r and r
are the
temperature- and pH-dependent anisotropies for free and bound states,
respectively(22) , which are calculated from polarization as r = 2P/(3 - P), where P is the polarization. The pH dependence of the Fl quantum yield was
corrected by incorporation of pH-dependent R values in . Dissociation constants (K
) at each
pressure were calculated using the following relation:
where [Abs] and [Fl]
were the total concentrations of Ab active sites for scFv, Fab,
or mAb and Fl, respectively(24, 25, 26) . The
dissociation constant related to the free energy of dissociation as
follows:
where R was the gas constant and T was the
temperature in Kelvin. The linear regions of the dissociation curves
were used to calculate values for K and
G
, for IgG, Fab, scFv 9-40/212, and scFv
9-40/212
at all temperatures and pH values.
V was obtained from the following relation:
Figure 1:
Iodide quenching of
fluorescence intensity of Fl bound to mAb 9-40 () and mAb
4-4-20 (
).
Figure 2:
Raw polarization data for dissociation of
Fl from scFv at 35 (), 25 (
), 15 (+), and 5 °C
(
) and IgG at 35 (
), 25 (
), 15 (
), and 5
°C (
).
Figure 3:
V (ml/mol) as a function of
temperature for mAb 9-40 (
), Fab 9-40 (
), and scFv 9-40
(
).
Figure 4:
Intensity recovery for Fl fluorescence
bound by mAb 9-40 (), Fab 9-40 (
), and scFv 9-40 (
)
as a function of pressure.
Figure 5:
Raw polarization data for dissociation of
Fl from IgG at pH 8.0 (), 7.5 (
), 7.0 (
), 6.5
(
), 6.0 (
), and 5.5 (+).
Figure 6:
V (ml/mol) as a function
of temperature for mAb 9-40 (
), scFv 9-40 (
), and scFv
9-40
(
).
Hydrostatic pressure has already been shown to be a valuable
tool in the study of protein subunit interactions because thermodynamic
information can be obtained under isothermal
conditions(22, 23) . The thermodynamic parameter of
interest in this study was V.
V and its
temperature dependence for mAb, Fab, and scFv 9-40/212 were found to be
identical within error (Fig. 3). Identity of the temperature
dependence for mAb 9-40 and its derivatives indicated that the change
in volume upon Fl dissociation occurred specifically in the variable
domains; in other words, no influence by constant domains was observed
under hydrostatic pressure. Values of
V obtained as a
function of pH are shown in Fig. 6. The pH dependence of
V demonstrated that an ionizable group specifically in
the active site was titrated in the pH range observed (Fig. 6).
A lack of pH dependence of
V for scFv
implied that the His in the native 9-40 active site was the
titratable group.
In order to fully understand the behavior of
V observed in this study, it is desirable to know which
amino acids are the ligand contact residues within the 9-40 active
site. The topographical structure of the 9-40 active site was not
available, but knowledge of the 4-4-20 active site provided important
information about the mAb 9-40 active site. Overall, the 4-4-20 active
site is an electropositive environment that complements the
electronegative influence of Fl, which is a dianion, at neutral pH, in
aqueous media. Fl is composed of two substructures: the xanthenone ring
and the phenyl carboxyl moiety, and it is known from the crystal
structure to be a site-filling antigen (18, 19) . In
4-4-20, the xanthenone ring occupies the deepest part of the Ab active
site and resides in an aromatic slot flanked by Tyr-32L, Trp-96L, and
Trp-33H. This aromatic-aromatic stacking stabilizes the xanthenone ring
in the active site by van der Waals' forces. The electrostatic
interactions with Fl in the 4-4-20 active site include the first enolic
oxygen salt bridged to the Arg-34L, the second enolic oxygen hydrogen
bonded to the His-27L, and the phenyl carboxylate oxygen hydrogen
bonded to Tyr-32L. In the 4-4-20 crystal structure, the charged Arg-34L
coordinates two water molecules as it salt links to the enolic oxygen
group.
Although the crystal structure of mAb 9-40 had not been
solved, the amino acid residues that comprised its active site were
inferred from the crystal structure of mAb 4-4-20. Wang et al.(28) have suggested three types of combining sites:
cavity, groove, and planar. This topographic classification is based on
the analysis of 20 Ab x-ray structures. Presumably the shape of the
active site of mAb 9-40, whose amino acid sequences of the variable
domains differ only by 10 residues (15) , should not change
significantly from mAb 4-4-20 pocket shape nor should the relative
positions of the residues. Based on this premise, Omelyanenko et
al.(29) used molecular modeling to show that the distance
of His-34L was 3.5 Å from the Fl enolic group so that sterically
no salt link could exist. The His-34L or the Fl enolic group was the
most likely source of this pH dependence of V, observed
in this study, because both His (30) and the Fl enolic oxygen (31) were found to be titrated in the pH range observed.
However, the hydrostatic pressure behavior of scFv
lended credence to the hypothesis that the pH dependence was due
to the ionization of the His-34L.
To discuss the temperature and pH
dependence of V in relation to the 9-40 active site, the
origin of
V should be understood. Several factors have
been proposed to contribute to the change in the partial molar volume
that accompanies ligand dissociation from a protein(22) :
replacement of interaction between nonpolar groups at two sides of the
apolar boundary by water dipoles, imperfect packing of the liganded
state of the Ab, and electrostriction of water by newly formed charged
groups. In the first such contribution to
V, replacement
of interactions between nonpolar groups at the two sides of the apolar
boundary by water dipoles, the apolar boundary of interest in our
system was that between Fl and the active site. Studies have shown that
water molecules form a three-dimensional network that bridges the
antigen with the Ab active site increasing the complementarity between
the interacting surfaces(32) . Iodide quenching of Fl bound by
mAb 9-40 demonstrated that Fl was solvent-accessible. The fact that Fl
was solvent accessible and that water molecules have been shown to be
present in Ab active sites, in general, make greater interactions of
nonpolar groups at the apolar boundary upon dissociation unlikely as a
major contribution to
V for mAb 9-40. A second
contribution to
V may have been the existence of free
volume due to imperfect packing. mAb 9-40 is the germ line clone of the
4-4-20 idiotype family. The mutated residues in mAb 4-4-20 active site
varied in volume significantly from the original germ line
residues(33) . Imperfect organization of volume in the original
germ line residues can cause the packing in mAb 9-40 to include
cavities or voids such as that observed by Omelyanenko et al.(29) at the His-34L site in 9-40 using molecular modeling.
The existence of cavities or voids would have provided additional
flexibility at the site of the void, which may have eventually
translated to an unstable environment for the Fl hapten and thus low
affinity. Accordingly, higher affinity would then result from somatic
mutations, which improved active site packing as previously suggested
by Herron et al.(34) . Higher temperatures could have
increased thermal energy of the solvent bath, which provided energy for
fluctuations in the active site that allowed migration of solvent to
fill these cavities. The temperature dependence would have been from
subsequent decrease of voids where the temperature was increased, which
would have diminished
V. The last determinant of
V was electrostriction of water by newly exposed charged
groups. The decrease in
V, which can arise from
electrostriction of water of
20 ml/mol or 30
Å
/molecule, is not negligible. Newly exposed charged
groups should not have occurred in the exterior of the IgG molecule
upon Fl dissociation because the degree of solvent exposure of the
electrostatic surface charge was not expected to change. Therefore,
newly exposed charge groups from Fl dissociation, if present, would
have occurred in the active site. The identity of the dissociation
behavior observed for the IgG, Fab, and scFv 9-40/212 provided
experimental evidence that localized
V to the variable
domains, probably in the active site.
In the 9-40 active site, the
34L position was occupied by a His. The salt link between His-34L and
the enolic oxygen was not expected to exist because the geometry of His
restricts its radial charge range. All else being equal, a void would
have existed where the salt link and the coordinated water molecules
appeared in the 4-4-20 crystal structure(18, 19) . The
titration of His-34L in the 9-40 active site may then have given rise
to pH dependence of V from the ionization of His in the
active site and coordination of water by charged His similar to those
observed in the crystal structure of 4-4-20. The initial premise was
that a cavity or void existed at the site of the His because of its
geometry with respect to Fl. Although it had been shown that water
molecules may line the active site and Fl was solvent-accessible, this
did not preclude the presence of voids deep in the active site. The
crystal structure of 4-4-20 showed that the 34L position lay deep in
the hydrophobic pocket of the active site and the residue at that
position in 9-40 should have followed suit. At high pH, the His should
have been neutral and could not have coordinated any water molecules at
that position thus leaving a void. The dissociation of Fl from the
active site would have contributed to
V if upon
dissociation the void was then filled by a water molecule. Protonation
of the His at low pH would have then imparted a positive charge on that
residue that allowed His to coordinate a water molecule at that
position. Occupation of the void by a coordinated water molecule would
have decreased
V upon Fl dissociation at lower pH because
of the lack of a void due to occupation by a coordinated water
molecule. The difference in
V observed as the pH
decreased from pH 8.0 to pH 5.5 was approximately 16 ml/mol or 24
Å
/molecule, which is comparable with the volume of a
water molecule. Accordingly, the curve in Fig. 6would then
suggest that the pK
of His is pH 7.4 or 1.2 pH
units greater than its aqueous value. It is well known that
pK
of individual residues vary significantly when
in a protein environment(35) . The other ionizable group was
that of the Fl enolic oxygen. The pK
of the enolic
oxygen of Fl in mAb 9-40 was found to be 6.7 by Omelyanenko et al.(29) , which was higher than the pK
for the enolic oxygen of free Fl at 6.2(31) . However,
both the bound and free pK
values of the enolic
oxygen of Fl were lower than the midpoint observed in Fig. 6.
Therefore, the pH dependence of
V was attributed to the
ionization of His-34L and the possible coordination of water molecules
at that site.
Denzin and Voss (10) found that mutation of
His-34L to Arg decreased the affinity of scFv 9-40/212, although it was
not possible to obtain a value. In this study using hydrostatic
pressure, the K for scFv
was
found to be 2.2
10
M
,
which was lower than the K
of the native molecule.
The pressure dependence of scFv 9-40/212
did not show
the same type of titration behavior as a function of pH in this range.
The value of
V remained constant at the pH range
observed, indicating that the site specific mutation mitigated the
titration of the ionizable group (Fig. 6). According to the
above hypothesis, this was an expected result because the Arg was
ionized throughout the pH range used in this study. The ionization of
Arg allowed the coordination of water molecules such as those seen in
the 4-4-20 crystal structure throughout the pH range observed.
Denzin and Voss (10) constructed scFv to
test the hypothesis that the salt link between Arg-34L and the enolic
oxygen was responsible for the high affinity of 4-4-20 as previously
had been suggested by the lower affinity of scFv 4-4-20/212
(9) . Investigators using x-ray crystallography have
previously shown that in certain Ab-antigen systems(32) , a
site-directed mutation only affected the area where the mutation
occurred. In that study, with the crystal structure of Fv D1.3, anti
hen lysozyme Ab, and its site directed mutant Fv
D1.3
, where the affinity of this Ab for its antigen
decreased 3-fold, it was found that all the detectable changes in
conformation occurred around the site of mutation. The rest of the
antigen-Ab interface remained as in the complex with the wild type Fv.
The fact that the mutation of His-34L to Arg caused a decrease in
affinity where an increase was expected indicated that the Arg salt
link was not the only determinant of 4-4-20 high affinity.
In an
effort to study the effect of constant domains on binding,
Müller et al.(36) investigated
the relative dynamics of the variable domains of mAb 4-4-20 with its
Fab 4-4-20 and scFv 4-4-20/212 derivatives. In that study, the variable
domains were identical, but the Abs differed in the amount of constant
domains. Müller et al.(36) found
that scFv 4-4-20/212 was substantially less stable than the Fab 4-4-20
and mAb 4-4-20. These results were obtained for mAb 4-4-20 and its
derivatives specifically as a glycerol-water mixture. Coelho-Sampiao
and Voss (26) studied scFv 4-4-20/212 with hydrostatic pressure
and found that V of dissociation for Fl-scFv 4-4-20/212
was 10 times higher than that found for Fl dissociation from intact mAb
4-4-20. The greater dissociation of Fl from scFv 4-4-20/212 was
explained in terms of a higher overall flexibility of unliganded scFv
4-4-20/212 and of a less stable binding site in scFv 4-4-20/212
relative to mAb 4-4-20. Although informative, these studies compared
derivatives that contained identical variable domains but possessed
slightly different affinities (mAb 4-4-20 K
= 1.3
10
M
; scFv 4-4-20/212 K
= 2.9
10
M
). In this study, mAb 9-40, and its
derivatives were used because the affinities are identical for all. It
was found that no difference in ligand dissociation behavior between
scFv 9-40/212 and mAb 9-40 was observed, which indicated that the
variable domains dynamics were not coupled to the constant domains for
mAb 9-40 and its derivatives in contrast to that observed for 4-4-20.
This result was originally unexpected given the lack of the
C
1 and the C
1 constant domains, which were
previously found to stabilize mAb 4-4-20(26, 36) .
However, this finding may be explained in terms of increased
conformational domain dynamics for 9-40 and may have been predicted by
the identical affinities for mAb 9-40 and its derivatives.
The
relationship of Ab structure and dynamics to affinity can be visualized
by a model originally proposed to predict different rates of
association for an Ab with cross-reactive ligands that has been
modified to include metatype dynamics (Fig. 7). The nonliganded
conformers correspond to a dynamic distribution of idiotypic states
similar to distribution of states discussed in Frauenfelder and
Wolynes(12) . A number of unliganded conformers in this
distribution can bind the homologous ligand. Once bound by the ligand
the width of the Ab-homologous-ligand-conformer distribution is
expected to decrease because the ligand stabilizes the Ab dynamics.
Cross-reactivity occurs when a conformer from the nonliganded conformer
distribution binds to a nonhomologous ligand and restricts the liganded
conformer distribution to an Ab-nonhomologous-ligand distribution
(represented by metatype distribution x or y) with average set of
structural determinants that probably differs from those held by the
Ab-homologous-ligand complex distribution (represented by metatype
distribution w). For 4-4-20, no cross-reactivity existed. It bound with
high affinity to Fl and a conformational change was known to occur once
the ligand was bound(16, 17) . For 9-40, the value for
V at pH 8.0, 25 °C was greater than that observed for
mAb 4-4-20. It has been shown in this study that a possible determinant
of
V for mAb 9-40 was the presence of voids or cavities.
Although the exact fractional contribution from the three causes of
V previously mentioned at present cannot be quantitated,
the more likely contribution to the difference in
V between mAb 9-40 and mAb 4-4-20 may have been imperfect packing
from the presence of voids. The presence of voids can contribute to the
distribution of states of 9-40 by migration throughout the Ab variable
domain matrix(37) . An ensemble of Ab molecules each with a
void in a slightly different position would be a distribution of
conformations (12) . As a result, the distribution of
nonliganded conformations for 9-40 may be greater than that for 4-4-20.
The presence of an unliganded distribution of states for mAb 9-40 was
corroborated by the ability of 9-40 to cross-react with Fl analogs (
)in contrast to 4-4-20. Moreover, the bound state of 9-40
was believed to have a wider distribution of conformations than 4-4-20,
as well. The overall distribution of ligand states for 9-40 would be
greater than 4-4-20 because the structural characteristics of the
observed cross-reactive species would not be expected to be the same.
This hypothesis was in agreement with the iodide quenching of bound Fl.
Increased dynamics of the 9-40 variable domains compared with 4-4-20
would have allowed Fl to be more accessible to the quencher. The higher
affinity of 4-4-20 can be viewed as a consequence of, among other
factors, a high degree of conformational rigidity, partly stabilized by
the constant domains, which translated to a smaller distribution of
nonliganded cross-reactive conformers. In summary, the dynamic
fluctuations of the 9-40 variable domains were believed to be below the
limit of flexibility that could be stabilized by constant domains.
Figure 7: Model depicting relationship between Ab stucture and dynamics.