Thermodynamic Analysis of the Increased Stability of Major
Histocompatibility Complex Class II Molecule
I-Ek Complexed with an Antigenic Peptide at an Acidic
pH*
Keigo
Saito
,
Akinori
Sarai§,
Masayuki
Oda
,
Takachika
Azuma
, and
Haruo
Kozono
¶
From the
Research Institute for Biological Sciences
(RIBS), Tokyo University of Science, 2669, Yamazaki, Noda, Chiba
278-0022, Japan and the § Tsukuba Institute, Institute of
Physical and Chemical Research (RIKEN), Tsukuba,
Ibaraki 305-0077, Japan
Received for publication, January 31, 2003
 |
ABSTRACT |
The differential scanning calorimetry analysis of
the murine major histocompatibility complex class II molecule,
I-Ek, in complex with an antigenic peptide derived
from mouse hemoglobin, showed that the thermal stability at the mildly
acidic pH is higher than that at the neutral pH. Although the thermal
unfolding of I-Ek-hemoglobin was irreversible, we extracted
the equilibrium thermodynamic parameters from the kinetically
controlled heat capacity curves. Both the denaturation temperatures and
the enthalpy changes were almost independent of the heating rate over
1 °C per min. The linear relation between the denaturation
temperature and the calorimetric enthalpy change provided the heat
capacity changes, which are classified into one for the mildly acidic
pH region and another for the neutral pH region. The equilibrium
thermodynamic parameters showed that the increased stability at the
mildly acidic pH is because of the entropic effect. These thermodynamic
data provided new insight into the current structural model of a
transition to an open conformation at the mildly acidic pH, which is
critical for the peptide exchange function of major histocompatibility complex class II in the endosome.
 |
INTRODUCTION |
The major histocompatibility complex
(MHC)1 class II is expressed
by professional antigen-presenting cells, which present peptide antigens to CD4 T cells. The newly synthesized MHC class II is transported from the endoplasmic reticulum to acidic compartments as
the complex with an invariant chain (Ii). In that complex, the peptide
binding groove of MHC class II is occupied by the CLIP, which is part
of Ii (1, 2). The antigenic peptides derived from endocytosed proteins,
which have been digested by cathepsins into 15-20-amino acid segments,
are then loaded onto the peptide-binding groove of the MHC class II in
exchange for the CLIP, at an endosomal pH (3-5). The role of the
acidic pH is considered to enhance the peptide exchange reaction rate
as well as to provide a suitable reaction condition for cathepsins. Finally, the peptide-MHC class II complex is transported from the
acidic compartments to the cell surface for the interaction with T cell
receptors (6).
The acidic pH can change the properties of MHC class II molecules and
accelerate the peptide exchange, because of the faster association
and/or dissociation reactions (7-9). To understand the
pH-dependent functions of MHC class II molecules, it is
essential to study their thermodynamic and structural properties.
Previous thermal stability analyses have determined the energetic
consequences of an alteration in the pH (10, 11). In the present study, we characterized the thermal stability of the murine MHC class II
molecule, I-Ek, in complex with the peptide, 64-76, of the
d allele of mouse hemoglobin (Hb), using differential scanning
calorimetry (DSC) measurements. As compared with other MHC class II
molecules, the peptide binding to I-Ek is most affected by
pH (7). The binding of the fluorescent dye
1-anilinonaphthalene-8-sulfonic acid, a probe for exposed nonpolar sites, to I-Ek was enhanced by an acidic pH,
indicating that the acidic environment is associated with an increase
in the exposed hydrophobicity in class II molecules (12, 13). The
analysis of the migration of I-Ek by SDS-PAGE showed that
the "floppy" form is observed at pH 4.5 in addition to the
"compact" form (14). The crystal structures of I-Ek-Hb
and its mutants have been determined, and the structural difference between the acidic and neutral pH values has also been discussed in
relation with the peptide exchange mechanism (11, 15, 16). Together
with the biochemical analyses, the conformational change from a
"closed" to an "open" form is considered to occur at low pH and
to be critical for the function. Thus, I-Ek may be a good
target for further thermodynamic characterizations and for studying
their relation to the structural features.
DSC analyses can provide accurate thermodynamic parameters, such as the
calorimetric enthalpy change (
Hcal) and the
entropy change (
S), in addition to the denaturation
temperature (Td) and the van't Hoff enthalpy change
(
HvH), which are also obtained in circular
dichroism (CD) measurements. It is interesting to determine the
energetic contribution to the increased stability of MHC class II
molecules at low pH relative to that at neutral pH (10), which can
explain the structural and functional differences. Additionally, to
analyze the irreversible thermal denaturation of I-Ek-Hb,
the kinetically controlled heat capacity functions were used for the
extraction of equilibrium parameters (17). Because reversible denaturation is limited to small, compact proteins, the evaluation of
irreversible transitions will be more important to analyze structure-function relationships in the present post-genome era. Under
the same conditions used for DSC measurements, with little interference
from the irreversible process, the thermodynamic parameters can be
quantitatively determined and compared as a function of pH.
 |
EXPERIMENTAL PROCEDURES |
Expression and Purification of MHC Class
II--
Baculovirus-infected insect cells, Sf9, were used to
produce soluble I-Ek molecules with the Hb peptide, 64-76,
linked to the N terminus of the
subunit via flexible linker, as
described previously (18, 19). The I-Ek-Hb molecule was
secreted into the medium and was purified by immunoaffinity column
chromatography with 14-4-4S, a monoclonal anti-MHC class II antibody,
followed by gel filtration chromatography using a Superdex 200 column
(16 mm × 60 cm, Amersham Biosciences). The purified
fractions were pooled, and the buffer was exchanged to an appropriate
buffer. The protein concentrations were determined from UV absorption
at 280 nm and were calculated by using an absorption coefficient of
1.23 cm2 mg
1, which was estimated from the
amino acid composition of I-Ek-Hb.
CD Measurements--
Far-UV CD spectra of I-Ek-Hb
were measured on a Jasco J-600 spectropolarimeter. The protein
concentration was 0.36 mg/ml, and the optical path length was 0.1 cm.
Spectra for CD between 200 and 250 nm were obtained in 10 mM phosphate buffer containing 150 mM NaCl at
20 °C using a scanning speed of 10 nm min
1, a time
response of 2 s, a band width of 1 nm, and an average over 5 scans.
DSC Measurements--
DSC experiments were carried out on a
Microcal MCS DSC calorimeter. All solutions (10 mM
phosphate buffer containing 150 mM NaCl or 10 mM MOPS buffer containing 150 mM NaCl) were
carefully degassed before the measurements. Data were collected in the
temperature range between 20 and 90 °C at various heating rates,
0.2, 1.0, 1.5, and 2.0 °C min
1. The protein
concentration was in the range from 0.08 to 0.43 mg ml
1.
The analyses were done by a non-linear least-square method, as
described in the DSC analysis software Microcal Origin version 4.1. The
Hcal was calculated by integrating the area
in each heat capacity curve. The van't Hoff enthalpy change
(
HvH) was calculated by the next equation for
assuming the two-state transition,
|
(Eq. 1)
|
where Cp is the molar excess heat capacity,
and R is the gas constant. Using the
Cp values with the
Hcal and Td values, the Gibbs free energy of unfolding as a function of temperature,
Gd(T),
could be calculated from the following equation.
|
(Eq. 2)
|
Determination of Activation Parameters--
A rate-limiting
irreversible unfolding is assumed to follow a reversible transition
(20).
|
(Eq. 3)
|
The temperature dependence of the first-order rate constant,
k2, is given by the Arrhenius equation,
|
(Eq. 4)
|
where Ea is the activation energy of
irreversible unfolding, and T* is the temperature at which
k2 is unity. A detailed theoretical analysis for
irreversible denaturation has been reported previously (17). The four
methods, A, B, C, and D, were applied to determine the activation
parameters for denaturation of I-Ek-Hb in this study.
Method A--
The rate constant, k2, is
obtained at each temperature from the next equation,
|
(Eq. 5)
|
where r is the heating rate in kelvin per second, and
H is the corresponding enthalpy change at a given
temperature. The activation energy is obtained from the Arrhenius
plot,
|
(Eq. 6)
|
where A = exp (Ea/RT*).
Method B--
The variation of Td with
r is given by the following equation.
|
(Eq. 7)
|
Method C--
The dependence of the enthalpy evolved with
temperature is expressed by the following equation.
|
(Eq. 8)
|
Method D--
The activation energy can be calculated from the
heat capacity at Td,
Cp(Td), by the following equation.
|
(Eq. 9)
|
 |
RESULTS |
CD Analysis of I-Ek-Hb as a Function of
pH--
To eliminate the binding of endogenous peptides to
I-Ek during its expression, the Hb peptide was attached by
a flexible linker to the N terminus of the I-Ek
subunit, and the soluble molecule, I-Ek-Hb, was purified as
a single peak on gel filtration high performance liquid chromatography
analysis, corresponding to the 
heterodimer (Fig.
1A). The SDS-PAGE analysis
also revealed the purity of the I-Ek-Hb molecule to be over
95%. The far-UV CD spectra of I-Ek-Hb were similar to
those of I-Ek, reported previously (12), and showed that
the secondary structure was not grossly altered as a function of pH
(Fig. 1B).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Gel filtration high performance liquid
chromatography and far-UV CD spectra of I-Ek-Hb.
A, a solution of I-Ek-Hb (10 µg) was loaded
onto a Superdex 200 column, and the elution was monitored at 280 nm.
The retention times of standard proteins to calibrate the column,
bovine serum albumin (68 kDa), hen egg albumin (45 kDa), and
chymotrypsinogen A (25 kDa), are also indicated. B, far-UV
spectra of I-Ek-Hb at pH 5.0, 5.5, 6.0. 6.5, 7.4, and 8.0 are superimposed. The vertical scale is normalized by the
mole concentration.
|
|
Thermal Denaturation Analysis of I-Ek-Hb as a
Function of pH--
For DSC measurements at mildly acidic and neutral
pH values, the I-Ek-Hb molecule was first dissolved in
phosphate buffer, because of its lower enthalpy change for the
deprotonation and its lower temperature dependence (21). Fig.
2 shows the excess heat capacity curves
as a function of pH, at a heating rate of 1 °C per min. All curves
were irreversible as shown by the lack of reproduced excess heat
capacity in the second scanning, as described below. The small
transition ranges of about 10 °C indicated the high cooperativity of
this transition. Assuming the two-state transition, the thermodynamic
parameters for denaturation of I-Ek-Hb were determined, and
are summarized in Table I. It should be
noted that the stability at the mildly acidic pH is higher than that at
the neutral pH. The correlation between Td and
Hcal could be classified into two groups, one
for the mildly acidic pH and the other for the neutral pH (Fig.
3). The heat capacity change
(
Cp), determined from this correlation, is 11.1 kJ mol
1 K
1 for the mildly acidic pH and
that of the neutral pH is 15.9 kJ mol
1
K
1.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Typical excess heat capacity curves of
I-Ek-Hb at pH 5.0, 5.5, 6.0. 6.5, 7.4, and 8.0, respectively. The temperature was increased by 1.0 °C per
min.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Thermodynamic parameters for denaturation of I-Ek-Hb as a
function of pH
The heating rate was 1.0 °C per min.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Correlation between
Td and
Hcal of I-Ek-Hb.
The Td and Hcal values
were taken from Table I. The slopes were determined by the linear least
square method for the mildly acidic and neutral pH regions,
respectively.
|
|
To evaluate the protonation effects on the buffer and the protein (22,
23), the thermodynamic stability of I-Ek-Hb, in a buffer
with a large heat of ionization, MOPS, was also analyzed using DSC.
Because the pH of MOPS buffer is largely dependent on the temperature
(21), the pH at the Td of the buffer used in this
study was determined and applied to compare the thermodynamics in the
phosphate buffer described above. Table
II summarizes the thermodynamic
parameters obtained in MOPS buffer. The thermal stability at an acidic
pH is higher than that at a neutral pH, which is similar to the
stability in the phosphate buffer. The
Hcal
values in MOPS buffer are relatively smaller than those in phosphate
buffer. This difference should be because of the protonation effects on
the buffer and the protein.
View this table:
[in this window]
[in a new window]
|
Table II
Thermodynamic parameters for denaturation of I-Ek-Hb in
MOPS buffer
The heating rate was 1.0 °C per min.
|
|
Dependence of the Protein Concentration on the Thermal Denaturation
of I-Ek-Hb--
To analyze whether the protein
concentration used in the present DSC measurements has an effect on the
thermal denaturation, mainly because of the irreversibility, the
thermodynamic parameters were determined as a function of concentration
(Table III). Within the range from 0.08 to 0.33 mg ml
1 at pH 7.4, both Td and
H values were independent of the protein
concentration.
View this table:
[in this window]
[in a new window]
|
Table III
Thermodynamic parameters for denaturation of I-Ek-Hb at pH 7.4 as a function of protein concentration
The heating rate was 1.0 °C per min.
|
|
Heating Rate Dependence on the Thermal Denaturation of
I-Ek-Hb--
To determine the temperature range in which
the unfolding of I-Ek-Hb is predominantly irreversible, the
heating was stopped at various temperatures and the solution was
subsequently cooled to 20 °C, followed by the next heating (Fig.
4). These heat capacity curves showed
that the irreversible step occurs early in the DSC scans at both pH 5.5 and 7.4. Thus, the calorimetric traces have to be analyzed according to
an irreversible model. We carried out DSC experiments at different
heating rates, 0.2, 1.0, 1.5, and 2.0 °C per min, at pH 5.5 and 7.4, to analyze the kinetically controlled denaturation of
I-Ek-Hb (Fig. 5). The
thermodynamic parameters obtained from each transition curve are
summarized in Table IV. Whereas the
Td value at the rate of 0.2 °C per min was
significantly lower than the others, those at the rate over 1 °C per
min were similar at both pH values, indicating that the transition
temperatures at high heating rates reach their maximum values with
little effect from the irreversibility. All of the
Hcal values obtained with the various heating
rates were similar, within experimental error.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
A series of repeated heating and cooling
steps of I-Ek-Hb at pH 5.5 (A) and 7.4 (B). A, the scans were stopped at 65 (1), 70 (2), 73 (3), 75 (4), and
80 °C (5). Curve 6 is the second scan after
the full scan, and curve 7 is the full scan, taken from Fig.
2. B, the scans were stopped at 50 (1), 55 (2), 60 (3), 63 (4), 65 (5), 70 (6), and 80 °C (7).
Curve 8 is the second scan after the full scan, and
curve 9 is the full scan, taken from Fig. 1. The heating
rate was 1.0 °C per min.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Variation with heating rate of
I-Ek-Hb unfolding at pH 5.5 (A) and 7.4 (B). The heating rate is indicated at each
transition curve. The protein concentration for the respective
measurements was 0.4 mg/ml.
|
|
View this table:
[in this window]
[in a new window]
|
Table IV
Thermodynamic parameters for denaturation of I-Ek-Hb at pH 5.5 and 7.4 as a function of heating rate
The protein concentration was in the range between 0.33 and 0.38 mg
ml 1.
|
|
Activation Parameters of Irreversible Denaturation of
I-Ek-Hb--
Sanchez-Ruiz et al. (17) proposed
four methods to evaluate the activation energy from the excess heat
capacity curves, using the scan rate dependence of irreversible
denaturation. The graphical presentations of the evaluations according
to methods A, B, and C, described under "Experimental Procedures,"
are shown in Fig. 6, and the analyzed
activation parameters, including method D, are summarized in Table
V. Although the activation energies
derived from the various methods differed, the average value was larger than the previously reported values for the denaturation of other proteins, which are in the range between 280 and 360 kJ
mol
1 (24). The large activation energy suggests that most
of the native structure of I-Ek-Hb should be denatured
before the irreversible transition occurs, and the thermodynamic
parameters for the denaturation of I-Ek-Hb could be
analyzed as an equilibrium quantity, as described above. Similar to the
activation energy, the T* values were almost independent of
the scan rate, and were higher than the Td values,
except for those from the scan rate of 2 °C per min (Tables IV and
V). Together with the results of the scan rate experiments, this result
supports the validity of the approximation to determine the equilibrium
thermodynamics from DSC experiments with the scan rate of 1 °C per
min.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 6.
Determination of activation parameters of
I-Ek-Hb unfolding at pH 5.5 (A-C) and 7.4 (D-F). A
and D, Arrhenius plots of lnk2
versus T 1, according to method A. B and E, plots of
ln(r/T )
versus T 1, according to method B. C and F, plots of
ln[ln( Hcal/ Hcal H)] versus T 1,
according to method C.
|
|
View this table:
[in this window]
[in a new window]
|
Table V
Activation parameters for denaturation of I-Ek-Hb at pH 5.5 and
7.4
The protein concentration was in the range between 0.33 and 0.38 mg
ml 1.
|
|
 |
DISCUSSION |
Irreversible thermal denaturation is observed in many proteins,
including MHC molecules, which makes it difficult to analyze the
precise thermodynamics. In the present study, we found the interesting
phenomenon of MHC stability as a function of pH, although the thermal
denaturation process was irreversible. The thermodynamic origin of the
increased stability at the mildly acidic pH could be because of the
dynamic properties of MHC molecules, which are important for the
function of MHC class II molecules. To determine the thermodynamics and
to evaluate their validity, the effects of the irreversibility should
first be analyzed under various conditions. Because the method to
extract thermodynamic equilibrium parameters from kinetically
controlled heat capacity curves has been applied successfully to
several systems (17, 24, 25), we used this method in the present study.
The Td and
H values were almost
independent at a protein concentration of around 0.3 mg
ml
1 and a heating rate over of 1 °C per min (Tables
III and IV). Additionally, the large activation energy and the high
T* value indicated that the thermal denaturation process
under these conditions could be analyzed as the equilibrium
thermodynamics, with little interference from the irreversible process
(Table V). Therefore, the thermodynamic parameters as a function of pH
(Table I), obtained with a heating rate of 1 °C per min and a
protein concentration of about 0.3 mg ml
1 are meaningful.
In the previous CD analyses of the thermal denaturation of MHC class II
molecules, the temperature was increased in a stepwise mode (10, 11).
Because it takes a few minutes at each temperature for equilibrium
attainment and recording, the Td values should be
lower than those obtained with the method of continuous heating used in
this study. This is supported by the fact that the stability of
I-Ek-Hb at pH 7.4 in the previous CD measurements was
similar to that at the heating rate of 0.2 °C per min in this study
(Table IV) (11). Under the conditions of a stepwise mode or a low
heating rate for the analysis of irreversible denaturation, the
Td value is largely dependent on the heating rate.
In the thermal denaturation process of I-Ek-Hb, three
transitions should be involved: 1) peptide dissociation from the MHC class II molecule, 2) dissociation of the 
heterodimer to each subunit, and 3) denaturation of each subunit. The excess heat capacity
curve of I-Ek-Hb should be the sum of these transitions.
Although the ratio
Hcal/
HvH under the
various conditions of the DSC measurements is around 0.9 to 1.4 (Tables
I, III, and IV), we cannot exclude the possibility of the existence of
intermediate states and/or the coupling of respective transitions.
Because the bound peptide contributes to the stability of MHC molecules
(26), the peptide dissociation should occur first, to cause the
subsequent denaturation. This is also supported by the results that the
thermal denaturation profiles of I-Ek in complex with
mutant peptides of Hb seemed to be cooperative, similar to that of
I-Ek-Hb, although their Td values
differed from each other.2
These results indicate that the apparent stability of the MHC-peptide complex is largely dependent on the binding kinetics and/or the affinity of the bound peptide. The empty MHC class II molecule, I-Ek without the peptide, showed apparent size
heterogeneity, including the 
heterodimer and the high molecular
weight aggregates, and the addition of peptide changed this
heterogeneous molecule into the homogeneous 
heterodimer (19,
27), indicating that the irreversible transition is followed by the
dissociation of each subunit and its denaturation.
Increased stability at a mildly acidic pH relative to that at a neutral
pH was also observed in a murine MHC class II molecule, I-Ab, in complex with CLIP and the antigenic peptide
derived from the
subunit of the I-E molecule, E
(28). In
contrast, the thermal stabilities of I-Ad-E
and
I-Ek complexed with the moth cytochrome c
peptide at a mildly acidic pH are similar to those at neutral pH (10).
Despite the stability difference at both pH regions, all of the MHC
class II molecules are resistant to a lower pH, that is in contrast to
the stability of the MHC class I molecule (10). The increased stability
at a mildly acidic pH of the other MHC class II molecules may also be
because of entropic effects, similar to I-Ek-Hb as
described below, which generally correlates with their functions in an
acidic compartment.
In addition to the difference in the Td values,
other thermodynamic parameters for denaturation at the mildly acidic pH
differed from those at the neutral pH. The correlation of
Td with
Hcal at various pH
values indicated that the folding of I-Ek-Hb could be
classified into two groups (Fig. 3), which is consistent with the
difference observed in the structural analyses, as described below. The
Cp values obtained from the slope of
Td versus
Hcal
in phosphate buffer were 11.1 kJ mol
1 K
1
for the mildly acidic pH and 15.9 kJ mol
1
K
1 for the neutral pH. To compare the obtained values
with those of other proteins with a similar size, the empirical method
proposed by Oobatake and Ooi (29) was applied and the
Cp value of a 420-residue (NR) protein was
calculated to be 30.2 kJ mol
1 K
1 from the
following equation.
|
(Eq. 10)
|
This value, 30.2 kJ mol
1 K
1, is larger
than the experimentally determined values of I-Ek-Hb at
either pH region, indicating that the surface of non-polar residues of
I-Ek-Hb is more accessible to the solvent than that of
other proteins. Furthermore, it should be noted that the
Cp value at the mildly acidic pH is lower than
that at the neutral pH. This is consistent with the previous results,
in which the acidification changed I-Ek into a more
fluctuating state with an increase in the exposed hydrophobicity, like
a molten globule state (12, 13).
The analyses of the protonation effects showed that the
Hcal values in the buffer with a larger heat
of ionization, MOPS, are smaller than those in the buffer with a lower
heat of ionization, phosphate, whereas the thermal stability at an
acidic pH is higher than that at a neutral pH in both buffers. The
difference of the
Hcal values should be
because of the enthalpy of buffer ionization and the linked protonation
effects (21, 22). Petrosian and Makhatadze (23)
successfully evaluated the contribution of linked protonation effects
on the stability of a 69-amino acid residue protein, with consideration
of the isoelectric point (pI). In the case of a 420-residue protein,
I-Ek-Hb, the pI value in the denatured state is estimated
to be 4.8 from the amino acid composition, and that in the native state is estimated to be 5.0 by the isoelectric focusing experiments (data
not shown). Amino acid residues such as Asp and His, with charges that
are affected at the pH region analyzed in this study, are thought to be
involved in the structural and functional characters of MHC class II
molecules (16, 30, 31). Therefore, the present thermodynamic results
obtained in phosphate buffer, which has lower enthalpy change for the
deprotonation, could be the comparable data to analyze the effects of
pH on the stability difference of I-Ek-Hb. Assuming the
enthalpy derived from linked protonation effects is compensated by the
large ionization enthalpy of MOPS buffer, as observed in the CspA
unfolding (23), the conformational enthalpy change can be estimated to
be about 700 kJ/mol. Still on this assumption, the increased stability
of I-Ek-Hb at acidic pH is because of a favorable
S, as described below.
To analyze the thermodynamic origin of the stability difference, the
thermodynamic parameters at 75.4 °C, the denaturation temperature at
pH 5.5, were calculated using the correlation of
G and
H with temperature (Table
VI). Within the narrow range of
temperature around the Td values, the errors of the calculated
G and
H values at the reference
temperature should be small, even if the
Cp
values used contain some errors, because of the temperature dependence
and the linked protonation effects. This result clearly indicates that
the higher stability at the mildly acidic pH than that at the neutral
pH is because of the difference in the entropic contributions. One
possible explanation for this entropic difference as a function of pH
is that the native structure of I-Ek-Hb at the mildly
acidic pH is more flexible, which can facilitate the peptide exchange.
This is consistent with the previous SDS-PAGE and structural analyses,
in which I-Ek gained flexibility at low pH (11, 14). The
smaller
Cp value of I-Ek-Hb at low
pH, as described above, should also be because of this increased
flexibility. Although the difference in the secondary and tertiary
structures between the mildly acidic and neutral pH values detected in
the CD measurements is subtle (Fig. 1B), similar to the
previous reports (12, 32), the molecule will be more dynamically
fluctuating and the surface of non-polar residues will be more
accessible to solvent at a mildly acidic pH.
View this table:
[in this window]
[in a new window]
|
Table VI
Thermodynamic parameters for denaturation of I-Ek-Hb in
phosphate buffer at the denaturation temperature at pH 5.5
|
|
The kinetic and structural analyses of I-Ek-Hb have shown
that the hydrogen bonding network, formed by the cluster of carboxylate groups around the P6 pocket, Asp66 and Glu11 of
the
subunit, Asp73 of the Hb peptide, and water
molecules, changes as a function of pH, which can regulate the
conformation of I-Ek and the peptide exchange rate (11).
The protonation of these carboxylate groups at low pH, together with
other residues such as His81 of the
subunit,
facilitates the flexibility and the conformational change of the MHC
class II molecule from the closed to the open form (30). In
addition, the recent mutational analyses of HLA-DR, the human homologue
of I-Ek, have shown that His33 of the
subunit has the role of a pH-sensitive switch at low pH, which can
regulate the conformational transition and the peptide exchange (31).
The decreased
H, T
S, and
Cp values for the thermal denaturation of
I-Ek-Hb at the mildly acidic pH relative to those at the
neutral pH should be because of the differences in the dynamic
properties, which are closely related with the function of MHC class II
molecules. The present DSC analyses provide thermodynamic insight into
the increased stability at the acidic pH.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Atsuko Yoshino and Dr. Koji
Furukawa, Tokyo University of Science, Dr. Haruki
Nakamura, Osaka University, Dr. Hatsuho Uedaira, RIKEN, Dr.
Shun-ichi Kidokoro, Nagaoka University of Technology, and Dr.
Harumi Fukada, University of Osaka Prefecture, for technical support
and helpful discussions. We gratefully acknowledge Dr. Motohisa
Oobatake, who died in August 2002, and pay tribute to a great scientist.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
81-4-7123-9937; Fax: 81-4-7124-1541; E-mail:
kozonoh@rs.noda.tus.ac.jp.
Published, JBC Papers in Press, February 9, 2003, DOI 10.1074/jbc.M301086200
2
K. Saito, A. Sarai, M. Oda, T. Azuma, and
H. Kozono, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
MHC, major
histocompatibility complex;
CLIP, class II associated invariant
chain-derived peptide;
Hb, hemoglobin;
DSC, differential scanning
calorimetry;
Hcal, calorimetric enthalpy
change;
S, entropy change;
Td, denaturation temperature;
MOPS, 3-(N-morpholino)propanesulfonic acid;
HvH, van't Hoff enthalpy change;
Cp, molar excess heat capacity;
Cp, molar excess heat capacity change;
Ea, activation energy of irreversible unfolding;
T*, temperature at which the rate constant from denatured to
irreversibly arrived state is unity;
Gd(T), Gibbs free energy of unfolding
as a function of temperature.
 |
REFERENCES |
1.
|
Cresswell, P.
(1994)
Annu. Rev. Immunol.
12,
259-293[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Cresswell, P.
(1996)
Cell
84,
505-507[Medline]
[Order article via Infotrieve]
|
3.
|
Mellman, I.,
Fuchs, R.,
and Helenius, A.
(1986)
Annu. Rev. Biochem.
55,
663-700[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Engelhard, V. H.
(1994)
Annu. Rev. Immunol.
12,
181-207[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Villadangos, J. A.,
and Ploegh, H. L.
(2000)
Immunity
12,
233-239[Medline]
[Order article via Infotrieve]
|
6.
|
Davis, M. M.,
Boniface, J. J.,
Reich, Z.,
Lyons, D.,
Hampl, J.,
Arden, B.,
and Chien, Y.
(1998)
Annu. Rev. Immunol.
16,
523-544[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Sette, A.,
Southwood, S.,
O'Sullivan, D.,
Gaeta, F. C.,
Sidney, J.,
and Grey, H. M.
(1992)
J. Immunol.
148,
844-851[Abstract/Free Full Text]
|
8.
|
Reay, P. A.,
Wettstein, D. A.,
and Davis, M. M.
(1992)
EMBO J.
11,
2829-2839[Abstract]
|
9.
|
Kasson, P. M.,
Rabinowitz, J. D.,
Schmitt, L.,
Davis, M. M.,
and McConnell, H. M.
(2000)
Biochemistry
39,
1048-1058[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Reich, Z.,
Altman, J. D.,
Boniface, J. J.,
Lyons, D. S.,
Kozono, H.,
Ogg, G.,
Morgan, C.,
and Davis, M. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2495-2500[Abstract/Free Full Text]
|
11.
|
Wilson, N.,
Fremont, D.,
Marrack, P.,
and Kappler, J.
(2001)
Immunity
14,
513-522[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Boniface, J. J.,
Lyons, D. S.,
Wettstein, D. A.,
Allbritton, N. L.,
and Davis, M. M.
(1996)
J. Exp. Med.
183,
119-126[Abstract]
|
13.
|
Runnels, H. A.,
Moore, J. C.,
and Jensen, P. E.
(1996)
J. Exp. Med.
183,
127-136[Abstract]
|
14.
|
Sadegh-Nasseri, S.,
and Germain, R. N.
(1991)
Nature
353,
167-170[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Fremont, D. H.,
Hendrickson, W. A.,
Marrack, P.,
and Kappler, J.
(1996)
Science
272,
1001-1004[Abstract]
|
16.
|
Kersh, G. J.,
Miley, M. J.,
Nelson, C. A.,
Grakoui, A.,
Horvath, S.,
Donermeyer, D. L.,
Kappler, J.,
Allen, P. M.,
and Fremont, D. H.
(2001)
J. Immunol.
166,
3345-3354[Abstract/Free Full Text]
|
17.
|
Sanchez-Ruiz, J. M.,
Lopez-Lacomba, J. L.,
Cortijo, M.,
and Mateo, P. L.
(1988)
Biochemistry
27,
1648-1652[Medline]
[Order article via Infotrieve]
|
18.
|
Kozono, H.,
Parker, D.,
White, J.,
Marrack, P.,
and Kappler, J.
(1995)
Immunity
3,
187-196[Medline]
[Order article via Infotrieve]
|
19.
|
Kozono, H.,
White, J.,
Clements, J.,
Marrack, P.,
and Kappler, J.
(1994)
Nature
369,
151-154[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Lumry, R.,
and Eyring, H.
(1954)
J. Phys. Chem.
58,
110-120
|
21.
|
Fukada, H.,
and Takahashi, K.
(1998)
Proteins
33,
159-166[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Baker, B. M.,
and Murphy, K. P.
(1996)
Biophys. J.
71,
2049-2055[Abstract]
|
23.
|
Petrosian, S. A.,
and Makhatadze, G. I.
(2000)
Protein Sci.
9,
387-394[Abstract]
|
24.
|
Vogl, T.,
Jatzke, C.,
Hinz, H. J.,
Benz, J.,
and Huber, R.
(1997)
Biochemistry
36,
1657-1668[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Freire, E.,
van Osdol, W. W.,
Mayorga, O. L.,
and Sanchez-Ruiz, J. M.
(1990)
Annu. Rev. Biophys. Biophys. Chem.
19,
159-188[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Simon, A.,
Dosztanyi, Z.,
Rajnavolgyi, E.,
and Simon, I.
(2000)
Biophys. J.
79,
2305-2313[Abstract/Free Full Text]
|
27.
|
Stern, L. J.,
and Wiley, D. C.
(1992)
Cell
68,
465-477[Medline]
[Order article via Infotrieve]
|
28.
|
Tobita, T.,
Oda, M.,
Morii, H.,
Kuroda, M.,
Yoshino, A.,
Azuma, T.,
and Kozono, H.
(2003)
Immunol. Lett.
85,
47-52[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Oobatake, M.,
and Ooi, T.
(1993)
Prog. Biophys. Mol. Biol.
59,
237-284[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
McFarland, B. J.,
Katz, J. F.,
Beeson, C.,
and Sant, A. J.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
9231-9236[Abstract/Free Full Text]
|
31.
|
Rötzschke, O.,
Lau, J. M.,
Hofstätter, M.,
Falk, K.,
and Strominger, J. L.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
16946-16950[Abstract/Free Full Text]
|
32.
|
Lee, J. M.,
Kay, C. M.,
and Watts, T. H.
(1992)
Int. Immunol.
4,
889-897[Abstract]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.