We constructed 18 single amino acid mutants of
the adipocyte fatty acid-binding protein (A-FABP) and 17 of the
intestinal fatty acid-binding protein (I-FABP), at locations in the
fatty acid (FA) binding sites. For each mutant protein, we measured thermodynamic parameters that characterize FA binding. Binding affinities ranged from about 200-fold smaller to 30-fold larger than
the wild type (WT) proteins. Thermodynamic parameters revealed that
binding affinities often inaccurately reported changes in protein-FA
interactions because changes in the binding entropy and enthalpy were
usually compensatory and larger than the binding free energy. FA-FABP
interactions were quite different for I-FABP and A-FABP proteins.
Binding affinities were larger and decreased to a greater degree with
increasing FA solubility for most of the I-FABP as compared with the
A-FABP proteins, consistent with a more hydrophobic binding site for
the I-FABP proteins. In A-FABP, Ala substitutions for
Arg106 and Arg126, which interact with
the FA carboxylate, reduce affinities by about 100-fold,
but in I-FABP, R106A increases affinities up to 30-fold.
Moreover, in A-FABP, the thermodynamic parameters predict that the FA
carboxylate location switches from the 126-position in R106A to the 106 position in R126A. Finally, the A-FABP proteins, in contrast to the
I-FABP proteins, reveal significant heat capacity changes
(
Cp) upon FA binding, and substitutions at
residues Arg106 and Arg126 reduce the magnitude
of
Cp.
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INTRODUCTION |
Fatty acid-binding proteins
(FABPs)1 are approximately
15-kDa cytosolic proteins that probably play important roles in fatty acid (FA) metabolism (1-4). FABPs have been found in a wide variety of
cells and form a family of proteins whose amino acid sequence identity
varies between about 25 and 95% (3). Although the amino acid sequences
of this family of proteins differ, their backbone structures are
virtually identical (3). X-ray crystallography and NMR studies indicate
that the dominant feature of the FABP structure is a "clam shell"
formed by 10 orthogonal
-strands (Fig. 1A). These studies
also reveal that the FA binds to a site that is internal to the protein
(5-11).
Although the three-dimensional structures of the proteins are quite
similar, the conformation of the FA within the binding site differs
considerably for the different members of this family. In particular,
in the mouse adipocyte (A-FABP) the FA conformation is curved relative
to the FA bound in the rat intestinal FABPs (I-FABPs), which shares
only about 25% sequence identity with the adipocyte protein
(Fig. 1A). Because of these FA
conformation and amino acid sequence differences, the amino acids
interacting with the FA within the binding cavity differ appreciably
for these two FABPs (Fig. 1, B and C). In the
adipocyte, the FA is within 4.5 Å of about 18 amino acid residues and
about 9 of these residues are hydrophobic, while in the intestinal
FABP, 19 amino acid residues are within 4.5 Å of the FA and about 14 are hydrophobic (12, 13).

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Fig. 1.
Oleate in the binding sites of the adipocyte
and intestinal FABPs. These images were generated using the x-ray
crystallographic coordinates for A-FABP and I-FABP determined in the
studies of Xu et al. (13) and Sacchettini et al.
(12), respectively. A, in this image, the backbone
structures of the holo forms of both proteins were merged using the RMS
overlay facility of the program HyperChem (Hypercube, Inc.,
Gainesville, FL). Oleates bound to A-FABP and I-FABP are shown together
with the A-FABP backbone. This image, as well as those in B
and C, was rendered using the program RasWin (written by R. Sayle). B, oleate bound within the A-FABP cavity is shown
together with the amino acid side chains of the WT protein for which
substitutions were made in this study. C, oleate bound
within the I-FABP cavity together with the residues for which
substitutions were made in this study. Single-letter amino acid codes
are shown.
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FA binding affinities for the adipocyte and intestinal FABP are in the
1-400 nM range, and thermodynamic measurements reveal that
binding to both proteins is predominately enthalpically driven (14,
15). Binding characteristics do, however, differ for the two proteins.
In particular, affinities for most FAs are larger for I-FABP than
A-FABP. Moreover, the change in binding enthalpy (
11 kcal/mol) is
virtually independent of FA type for I-FABP, but it decreases
monotonically from
5 to
12 kcal/mol, with increasing FA solubility
from arachidonate to palmitate, for A-FABP.
To understand how the interactions between the FA and amino acids
within the binding cavity of I-FABP are related to binding affinities,
we have investigated the binding thermodynamics for FA binding to
single amino acid mutants of I-FABP, in which the single amino acid
substitutions were generated for most of the amino acid residues that
form the binding cavity (16). Results of these studies revealed that
binding affinities alone do not accurately reflect the change in the
underlying molecular interactions caused by the amino acid
substitution. A more accurate understanding of the interactions is
obtained by measurements of the change in binding enthalpy and entropy
induced by each mutant. In the present study, we have extended this
investigation to the adipocyte protein and several additional I-FABP
mutants both to understand the interactions in A-FABP and to attempt to
relate the differences in FA conformation and binding affinities in
these two proteins.
In particular, we have investigated the effect of single amino acid
substitutions at 15 sites within the FA binding cavity of A-FABP. We
measured binding affinities for FA to each mutant as a function of
temperature and used van't Hoff analysis to obtain the thermodynamic
parameters of binding. These results revealed enthalpy-entropy
compensation effects in the adipocyte proteins as observed previously
for mutants of the intestinal protein, and in addition, mutants of the
adipocyte protein reveal substantial changes in heat capacity for
single amino acid substitutions.
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EXPERIMENTAL PROCEDURES |
Materials--
All measurements were done, as described
previously (14, 15, 17), using the sodium salts of the FA purchased
from Nu Chek Prep (Elysian, MN). All binding measurements were done in a buffer consisting of 10 mM HEPES, 150 mM
NaCl, 5 mM KCl, and 1 mM NaHPO4, at
pH 7.4. The fluorescent free fatty acid probe, ADIFAB (acrylodated
I-FABP), was prepared from acrylodan-derivatized recombinant rat
intestinal fatty acid-binding protein (rI-FABP) as described (17) and
is available from Molecular Probes (Eugene, OR).
Mutants were constructed by extension of overlapping oligonucleotides,
which together spanned restriction endonuclease sites in I-FABP, and
insertion of the resulting double-stranded DNA as described (16, 18).
Mutant and WT proteins were expressed in the pET/BL21 system (Novagen,
Madison, WI) and purified as described previously (14, 15, 17). Five of
20 A-FABP mutants produced inclusion bodies (Y19A, D76A, R78A, I104A,
and Y128A), and of these, soluble protein (between 14 and 90 mg) was
recovered for Y19A, D76A, and R78A by solubilizing the inclusion bodies in 4 M guanidine HCl (GdnHCl) and then renaturing by
dialysis against a Tris buffer as described (16). The Y128A
precipitated from solution at 37 °C during Lipidex delipidation and
consequently binding was not measured for this protein. The I104A
mutant precipitated from solution upon renaturing the protein from
GdnHCl and therefore binding was not measured for this protein as well.
Although R78A and D76A also precipitated from solution, this occurred
at 45 and 50 °C, respectively; therefore, measurements were done
with these proteins. In general, I-FABP mutants had fewer problems of
recovery and higher yields than the A-FABP mutants.
After isolation, the proteins were stored at 4 °C in buffer
consisting of 50 mM Tris, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.05% sodium azide
at pH 8.0. The I-FABP mutants were found to be stable; binding
affinities for the I-FABP mutants were unchanged after many months of
storage. However, the A-FABP mutant proteins were much less stable.
Certain of the mutants (in particular M20A and M40A) showed large
decreases in binding affinity over a few months after purification,
indicating a general refolding or denaturation of the protein.
Moreover, the V115A mutant, although revealing a constant affinity,
showed a decrease in stoichiometry (from 1 to less than 0.3) over a
period of several months, suggesting a loss of protein through
denaturation. The results presented here were obtained using protein
within a few weeks of purification.
Fatty Acid Binding to FABP--
Measurements of the binding of
FA to FABP were done by using ADIFAB fluorescence to monitor the
binding of the sodium salts of the FA to each FABP at temperatures
between 10 and 45 °C as described (15). For both A-FABP and I-FABP
mutants, the binding affinities were measured for six FA (PA, SA, OA,
LA, AA, and LNA) at 37 °C. For I-FABP, the affinities for four FA
(PA, OA, LA, and AA) were measured from 10 to 45 °C. Binding
isotherms for I-FABP exhibited linear van't Hoff behavior, and
H0 values were determined from the slopes of
each of the van't Hoff plots.2
G0 values were evaluated from the
Kd values measured at 25 °C, and
T
S0 was calculated as
G0
H0. For the
A-FABP mutants, the affinities of OA and AA were measured at
temperatures ranging from 10 to 50 °C. The isotherms for the A-FABP
proteins did not in general reveal linear van't Hoff behavior, and
these data were analyzed using the integral form of the van't Hoff
expression to obtain values for
H0 and
T
S0 as well as for
Cp0 (19).
FABP Stability--
The conformational stability of I-FABP and
A-FABP WT and mutants proteins to denaturation by GdnHCl was determined
by measuring tryptophan fluorescence (
ext = 290 nm;
emis = 340 nm) with increasing concentrations of GdnHCl.
These results were analyzed assuming a two-state model using the
methods described previously for FABPs (20, 21). Thus, for each curve,
the intensities at 0 and 3 M GdnHCl were assumed to
represent the completely native protein and denatured proteins,
respectively. These values were used to calculate the ratio of
denatured to native protein (K) and therefore
G, from
G = RTlnK, at each GdnHCl concentration. Furthermore, we determined from the denaturation curves the concentration of GdnHCl
at which fluorescence was reduced by 50%
([D]50), the slope (mG) of
the variation of
G with [GdnHCl], and the free energy
difference (
Gapp) between the native and
denatured states extrapolated to zero [GdnHCl].
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RESULTS |
Binding Affinities of Mutant A-FABPs and I-FABPs--
We
engineered Ala substitutions at 15 A-FABP locations that are within 4.5 Å of the FA (Fig. 1B). In addition, we generated Gln
substitutions at residue positions 106 and 126 and a Gly substitution at position 57. We also engineered Ala substitutions in I-FABP at
positions analogous to 12 of the 15 locations of the adipocyte protein
(Fig. 1C). In addition, we substituted Gln at 106 and 126, Gly at 55, and Cys and Phe at 27. Results for nine of the 12 Ala
(positions 14, 17, 18, 23, 55, 115, 117, 106, and 126) and one of the
Gln (position 106) substitutions in I-FABP were reported previously
(16) and are reproduced here for comparison.
For each of these mutants, we measured binding affinities for several
long chain FAs at 37 °C (Table I)
using the ADIFAB method as described previously (15, 16, 19). The
results of these measurements are also shown as the differences
in the free energy of binding between the
mutant and WT proteins (
G0) in Figs. 2 and
3. The results for A-FABP reveal that,
relative to the WT protein, the single amino acid substitutions result in affinities that are virtually unchanged to ones that range from 50%
(V32A) to about 150-fold (Y19A and R126Q) smaller than the WT. In
contrast, the I-FABP mutants reveal a number of substitutions that
increase as well as decrease binding affinities so that mutant affinities range from about 30-fold lower
(
G0 = +2 kcal/mol) to 30-fold higher
(
G0 =
2 kcal/mol) than the WT protein.
These differences are particularly evident for the Ala substitutions at
positions 106 and 117 that result in lower affinities for the adipocyte
but higher ones for the intestinal proteins.
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Table I
Dissociation constants for I-FABP and A-FABP proteins
Kd values (in nM) were measured at
37 °C with uncertainties (S.D.) about 10%, as described previously
(14).
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Fig. 2.
Free energy differences for FA binding
between mutant and WT A-FABP proteins. Values of
 G0 = G0 (mutant) G0 (WT) in kcal/mol were determined at
37 °C for FA binding to the adipocyte proteins.
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Fig. 3.
Free energy differences for FA binding
between mutant and WT I-FABP proteins. Values of
 G0 = G0 (mutant) G0 (WT) in kcal/mol, were determined at
37 °C for FA binding to the intestinal proteins.
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Binding Thermodynamics of A-FABP and I-FABP Mutants--
Because
mutations will generally change both enthalpy and entropy and these
changes tend to compensate, changes in binding affinities may not
accurately reflect the effect that mutations have on the interactions
between FA and amino acid residues within the binding site (16, 22). We
have therefore carried out measurements of the binding affinities for
each of the WT and mutant proteins as a function of temperature and
have used the differential or integral form of the van't Hoff
expression to determine the thermodynamic parameters of the binding
reaction (see "Experimental Procedures" and Refs. 16 and 19). We
have previously reported that the temperature dependence for the WT
adipocyte protein was consistent with the differential form of the
van't Hoff expression, implying no change in heat capacity
(
Cp) upon ligand binding (15). However, more
extensive study of the binding characteristics of the adipocyte
proteins reveals a curvature in their van't Hoff plots that is
distinct from the linear behavior of the intestinal proteins (Fig.
4). We have therefore analyzed the WT and
mutant forms of the adipocyte proteins with the integral form of the van't Hoff expression to obtain the heat capacity change upon binding
as well as the enthalpy. The lack of significant heat capacity changes
observed for the WT form of the intestinal protein are consistent with
calorimetry measurements (23).

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Fig. 4.
Temperature dependence of oleate binding to
adipocyte and intestinal FABP mutants. These representative van't
Hoff plots for oleate binding to A-FABP and I-FABP proteins illustrate the linear and nonlinear behavior of binding for the I-FABP and A-FABP
proteins, respectively. The solid lines through the data represent fits using the differential and integral forms of the van't
Hoff equations and yield values of Cp = 620 kcal/mol/K and Cp = 0 kcal/mol/K for the V115A of
A-FABP and the Q115A of I-FABP mutants, respectively.
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Results at 25 °C for the differences in enthalpy
(
H0), entropy
(T
S0), free energy
(
G0), and heat capacity
(
Cp0) of oleate and
arachidonate binding between each mutant and the WT adipocyte protein
have been arranged in Fig. 5 so that the 
G0 values increase monotonically. The
results demonstrate that changes in affinities
(
G0) are not correlated with

H0,
T
S0, or

Cp0 because
|
H0| and
|T
S0| are large, relative
to |
G0|, and tend to compensate in
these binding reactions. Results for the I-FABP proteins also reveal
enthalpy-entropy compensation and a lack of correlation of these
changes with 
G0 (Fig.
6). These results include for I-FABP the
Ala substitutions for Lys27, Arg56, and
Asp74 as well as K27F, F55G, and R126Q, in addition to
those discussed previously (16). Although the overall patterns of
enthalpy and entropy changes are similar for the adipocyte and
intestinal proteins, the intestinal proteins are all consistent with
zero changes in heat capacity. In addition, the magnitude of
|
H0| and
|T
S0| for the intestinal
proteins are generally greater (average of 4 kcal/mol) than the
adipocyte proteins (average of 2 kcal/mol), but at the same time the
|
G0| values for the adipocyte are
equal to or greater than those for the intestine, implying weaker
interactions and a smaller degree of enthalpy-entropy compensation in
the adipocyte than the intestinal proteins.

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Fig. 5.
Mutant-WT changes in thermodynamic parameters
for A-FABP proteins. Values for  G0,
 H0,
T S0, and
 Cp0 were calculated as
Gmut0 GWT0,
Hmut0 HWT0,
T Smut0 T SWT0, and
Cp mut0 Cp
WT0, respectively.
G0, H0,
T S0, and
Cp0 ( Cp
WT0 = 0.5 for OA and 0.6 for AA) were
determined at 25 °C for oleate and arachidonate and are in units of
kcal/mol and kcal/mol/K, respectively. The results in this
figure have been arbitrarily arranged so that
 G0 values for oleate increase
monotonically. The scale for  G0 is
amplified 3-4-fold relative to those for
 H0, and
T S0. Negative values for
 H0 and
T S0 indicate more favorable
binding. S.D. values (in kcal/mol) are approximately 0.1-0.2 for
 G0 and 1-2 for
 Ho (indicated) and
T S0, and values are indicated
for  Cp0. *, not done.
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Fig. 6.
Mutant-WT changes in thermodynamic parameters
for I-FABP proteins. Values for  G0,
 H0, and
T S0 were calculated as in Fig.
5. G0, H0, and
T S0 were determined at 25 °C
for PA, OA, LA, and AA and are in units of kcal/mol. The results in
this figure have been arbitrarily arranged so that
 G0 values for linoleate increase
monotonically. The scale for  G0 is
amplified 2-3-fold relative to those for
 H0 and
T S0. Negative values for all
three parameters indicate more favorable binding. S.D. values (in
kcal/mol) are approximately 0.1-0.2 for  G0 and 1-2 for
 H0 and
T S0.
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Protein Stability--
Protein stability of the adipocyte and
intestinal WT and mutant proteins was assessed by the
GdnHCl-induced changes in intrinsic tryptophan fluorescence. Results shown in Fig. 7 and in Table II reveal that the half-concentration for
denaturation ranges between 0.8 and 1.6 M and that WT
A-FABP is somewhat more stable than WT I-FABP. Also shown in the table
are the mutant-WT differences in free energy change for denaturation. A
plot of these values versus the

G0 for binding reveals that
mutation-induced changes in stability and binding affinity are not
correlated (Fig. 8). This lack of stability-binding affinity correlation is consistent with results for
DNA binding and stability of the gene V protein of bacteriophage f1
(24).

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Fig. 7.
Guanidine HCl-mediated denaturation of A-FABP
and I-FABP proteins. Measurements shown are representative of
those that were used to obtain the results listed in Table II. In these
measurements, the tryptophan fluorescence ( ext = 290 nm,
emiss = 340 nm) was measured at 37 °C for the
indicated proteins, each at a concentration of 5 µM, as a
function of increasing concentrations of GdnHCl. Tryptophan intensities
were all normalized to the values obtained at zero GdnHCl.
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Table II
Stability of I-FABP and A-FABP proteins
Measurements of the WT proteins were done three times and once for each
of the mutants.
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Fig. 8.
Binding and denaturation are not
correlated. Values for  G denaturation for the
A-FABP and I-FABP proteins are from Table II, and the
 G0 values are data from Figs. 2 and 3. The
linear fit through this data has a correlation coefficient
(r2) of 0.0001.
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DISCUSSION |
In this study, we constructed site-specific mutants of the mouse
adipocyte- and rat intestinal fatty acid-binding proteins and measured
the temperature dependence of their binding to long chain fatty acids.
Single amino acid substitutions produced proteins that had binding
affinities ranging from about 200-fold smaller to 30-fold larger than
WT. Although affinities of the 17 intestinal FABP variants revealed
decreases as well as increases, the 18 A-FABP variants had affinities
that were either equal to or less than the WT. The thermodynamic
parameters for both sets of proteins revealed that binding affinities
often inaccurately reported changes in protein-FA interactions because
changes in the binding entropy and enthalpy were large and usually
compensatory, as described previously for I-FABP (16). Moreover, the
A-FABP proteins, in contrast to those from I-FABP, reveal significant
heat capacity changes upon FA binding, and the magnitude of
Cp was altered by specific single amino acid
substitutions.
Comparison of General Features of the FA Binding Characteristics of
Variants of the Adipocyte and Intestinal FABPs--
Binding affinities
of FA for FABPs reflect FA-water interactions involved in the
desolvation step and FA interactions with amino acids and bound water
within the FABP binding cavities. In the desolvation step, the
hydrophobic effect drives FA out of the water phase and into the FABP,
yielding a favorable contribution to the free energy of binding of
about
10 kcal/mol that is primarily entropic (15). Despite this large
and favorable entropic contribution to the binding free energy, the
binding free energy is almost entirely enthalpic, because the FA-FABP
interaction within the binding cavity involves a favorable enthalpy but
a nearly compensating unfavorable entropy change of about +10 kcal/mol
(15). Relatively small mismatches in these nearly compensatory
contributions to the free energy give rise separately to the observed
FABP specificity for different FA and to the magnitude of the binding
affinity. Although certain amino acid substitutions (e.g.
F17A, R106A or R106Q, Y117A, and R126A in I-FABP and V25A, R106A, and
C117A in A-FABP) change the specificity of FA binding (Table
III), virtually all of the amino acid
substitutions in I-FABP or A-FABP yield proteins that reveal increases
in affinity with decreasing FA solubility, indicating the importance of
aqueous solubility in determining the specificity of FA binding to
FABPs.
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Table III
Summary of thermodynamic parameter changes in Ala mutants of adipocyte
and intestinal FABPs
Values are for oleate binding to Ala mutants of A-FABP and I-FABP at
25 °C.
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Although solubility plays a major role in determining FA specificity
for both adipocyte and intestine, the specificity for different FA is
quite different for the two sets of proteins. The ratio of the
dissociation constant for a given FA relative to that for stearate
(Kd/Kd (SA),
provides a measure of the variation of binding specificity with FA
solubility (14). This ratio increases markedly with FA solubility for
most of the I-FABP mutants but to a much lesser degree for the A-FABP mutants, indicating that interactions within the binding cavity can, at
least partially, alter binding specificity (Fig.
9). As a consequence, the affinity for
polyunsaturated FA is much lower than for saturated FA of the same
chain length; on average, for all 17 I-FABP mutants, the affinity for
stearate (18:0) is 27-fold higher than for linolenate (18:3), and this
ratio of stearate to linolenate affinity is 10-fold smaller for the
adipocyte proteins (Fig. 9). Moreover, for the WT proteins, the
variation of Kd with FA solubility for I-FABP is
virtually identical to that expected for the transfer of FA from water
into a hydrocarbon solvent (slope = 1), while the results for
A-FABP are consistent with a more polar binding site (Fig.
10). Although single amino acid
substitutions can reduce the specificity for binding to I-FABP proteins
significantly, the A-FABP substitutions result in little reduction in
specificity because the WT protein reveals relatively little
specificity. In addition, although several A-FABP proteins (WT and
variants M40A, V115A, and K58A) display higher affinities for
polyunsaturated FA than WT I-FABP, none bind saturated and
monounsaturated FA as well as I-FABP. Thus, I-FABP may be designed for
maximum and A-FABP for minimum specificity differences. Indeed,
describing FA partition between aqueous solvent and lipid bilayers as a
binding process so that Kp
1/Kd (25), the Kd for stearate
binding to I-FABP is similar to that for partition into a hydrocarbon
solvent. Thus, as suggested by Fig. 10, A-FABP may achieve its more
uniform specificity for FA by being more water-like and thereby
providing less favorable interactions for saturated and monounsaturated
FA than I-FABP rather than by increasing its affinities for the
polyunsaturated FA. Although specific interactions can modify this
behavior, this trend of greater binding specificity with increasing
hydrophobicity of the binding cavity is consistent with the variation
observed previously among different WT FABPs (14).

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Fig. 9.
Variation of dissociation constants of I-FABP
and A-FABP proteins with FA solubility. The ratio of
Kd values for each FA to the values for stearate,
which has the lowest solubility, are plotted with increasing FA
solubility for each of the proteins of Table I.
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Fig. 10.
The relationship between the free energies
of FABP binding and water/membrane partition. Binding free
energies ( Gbinding0) for the
WT adipocyte and intestinal FABPs are from this study, and the
Gw-memb for the transfer from water to
membrane are from measurements of the partition into lipid vesicles
determined previously (15). The linear fit to the I-FABP data yields a
slope of 1.1 ± 0.1 and 0.3 ± 0.1 for A-FABP. The FA
corresponding to each
Gbinding0,
Gw-memb pair are indicated and abbreviated as
in Fig. 2.
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One of the most striking differences in the binding thermodynamics
between adipocyte and intestine is that binding to the adipocyte but
not the intestinal proteins involves a significant decrease in heat
capacity. The origin of this change is not due to FA-solvent water
interactions (e.g. to a reduction in the water-accessible area of the ligand (26)), because the heat capacity decrease is
FABP-specific. Indeed, |
Cp| is even larger
for liver than adipocyte FABP, and moreover, non-zero
Cp values were only observed for binding to one
(high affinity) of the two liver FABP binding sites (19). Thus,
Cp decreases must reflect specific alterations within the
adipocyte FABP binding site. Decreases in
Cp have
been associated with the reduction of accessibility to water of
nonpolar amino acid residues, and in particular, negative heat capacity
changes associated with ligand-protein interactions have been ascribed
to the decrease in accessible area of nonpolar residues (27). Unlike
the binding of ligands to sites on or near the protein surface, the FA
binding site is located in a cavity buried deeply within the protein
(2, 3). This binding site in both intestine and adipocyte FABP does,
however, contain a number of ordered water molecules in both the apo
and holo forms of the proteins, and more water molecules than are
revealed by x-ray diffraction may be present (3). Thus, binding of FA
within the FABP cavity may involve alterations in the accessibility of
internal nonpolar amino acid residues to water, presumably by FA
shielding of such nonpolar residues in A-FABP but not I-FABP. The
origin of this anomalous heat capacity of A-FABP is, however, more
complex than in the case of surface residues, because I-FABP, for which
Cp
0, has a larger number of nonpolar
residues within the cavity than A-FABP (3).
Results of the mutation studies may provide some clues to the origin of
Cp. To within the fairly large uncertainties in estimating
Cp, the results of Fig. 5 indicate that although most
mutations have relatively little affect on
Cp, both A and Q mutations at Arg106 and Arg126
reduce the magnitude of
Cp significantly. Water molecules at
the carboxylate end of the FA may be more structured in A-FABP than in
I-FABP. A-FABP has 10 bound waters, many of which are hydrogen-bonded
together to form a network that is more complex than the fairly linear
array of eight bound waters in I-FABP (9, 28). This network of hydrogen
bonds is also connected to Arg106, suggesting that water
structures involving this residue may be responsible for the anomalous
heat capacity of the adipocyte protein and that binding of FA
carboxylate disrupts this water structure.
Comparison of Specific Mutants of the Adipocyte and Intestinal
FABPs--
The portion of the binding free energy due to interactions
between the FA and amino acid residues and bound water within the FABP
reflects a sum of many contributions, certain ones of which may be
especially important for a given FABP and FA (2, 3, 16, 29). Here we
discuss the contribution of each amino acid substitution, focusing on
how these interactions differ in the adipocyte and intestinal proteins.
Although the three-dimensional structures of the adipocyte and
intestinal FABP backbones are virtually identical, the conformation of
the bound FA is very different in the two proteins (Fig.
1A). This difference in conformation and binding
characteristics reflects the different amino acid sequences of the
adipocyte and intestinal FABP. Investigating the effect of amino acid
substitutions of the residues directly involved in the FA-amino acid
interactions should therefore provide insight into the origin of the
differences in FA binding of the WT adipocyte and I-FABP proteins.
However, because the WT adipocyte and intestinal FABPs have different
amino acid sequences and different conformations of bound FA, the two
proteins have no exactly equivalent amino acid side chain-FA contacts
(Fig. 1). Nevertheless, as Fig. 1 also indicates, many of the amino
acid side chains occupy roughly equivalent positions relative to the FA
in the two proteins, and we will therefore compare the result of each
substitution made in the adipocyte protein with the equivalent one for
I-FABP. We will first discuss in detail the results for
Arg106 and Arg126, because mutagenesis of these
residues clearly illustrates how determination of the binding
enthalpies and entropies can provide greater insight about the binding
mechanism. The remaining amino acid residue-FA interactions will be
discussed according to whether mutants of the adipocyte and intestinal
proteins have no, similar, or different effects on the binding
affinities, as indicated qualitatively in Table III. (The sequence
alignment results in numbering for A-FABP that is 2 ahead of I-FABP
until residue 100 is reached, where the numbering is the same for both
proteins.)
Interactions with the FA Carboxylate--
Arginines at positions
106 and 126, which interact with the carboxylate end of the FA in the
adipocyte protein, appear to play important roles in all of the FABPs
that have been characterized (2, 3). Consistent with this expectation,
Ala and Gln substitutions at either the 106 or the 126 positions of the
adipocyte protein result in reductions in affinities (at 37 °C) of
between 7- and 110-fold. The large reduction in the R126Q affinity for
the FAs of Table I is consistent with previous results of Sha et
al. (30) for binding of paranaric acid to the adipocyte R126Q
glutathione S-transferase fusion protein. In contrast to
this expected behavior, substitutions for Arg106 in I-FABP
either have no effect (R106Q) or increase affinities by
about 30-fold (R106A). As described previously, I-FABP affinities are
either unchanged or increased upon substitution for Arg106,
because increases in entropy either equal or exceed decreases in
|
H| (16). These increases in entropy are consistent
with crystallography and NMR results that indicate greater mobility of
the FA in these mutants (29, 31). Although Ala and Gln substitutions
for Arg126 in I-FABP result in proteins with significantly
lower affinities (between about 20- and 150-fold), this is not due to a
direct interaction with the FA carboxylate, which is more than 10 Å from R126. As explained previously, the origin of the
Arg126 changes in I-FABP are probably due to the disabling
of an Arg126-Asp34 salt bridge (16).
The reductions in affinity (at 37 °C) observed with amino acid
substitutions at the 106- and 126-positions of the adipocyte protein
might be expected simply because favorable electrostatic interactions
are eliminated by these mutations. However, the interactions that
generate these changes are more complex, reflecting different enthalpy-entropy compensation and heat capacity changes for each substitution and FA. For example, at the 106 position, the affinity changes at 25 °C are due to a 3-4 kcal/mol less favorable
H and a smaller increase in entropy for R106Q but a 6 kcal/mol more favorable
H together with a
partially compensating reduction in entropy for R106A. In the case of
the 126-position, the large reductions in A-FABP affinity are produced
by relatively small decreases in |
H| (3-4 kcal/mol)
and almost no compensating entropy change for R126Q, to small (0-2
kcal/mol) |
H| increases for R126A and more
than compensating decreases in entropy.
These changes in the thermodynamic parameters for the 106 and 126 mutants of the adipocyte protein provide insights into the nature of
the interactions that generate the reductions in affinity and suggest
how the conformation of the FA may be altered by these mutations. Thus,
compared with WT, Ala substitutions for Arg106 and
Arg126 both result in more favorable enthalpies (at least
for oleate) but less favorable entropies (Fig. 5 and Table III). This
suggests that the strength of the electrostatic/hydrogen bond between
the FA carboxylate and the arginine's guanidinium group is increased and that, correspondingly, the disorder of the FA-Arg complex is
decreased in each of these mutants. Thus, in R106A the FA carboxylate should be more localized to Arg126 than in WT, and in
R126A, the carboxylate should be more localized to Arg106.
This reorientation may not occur for Gln substitutions, because, although the interaction is reduced relative to Arg, some degree of
hydrogen bonding between the carboxylate and Gln is retained. Thus, the
FA may be able to switch from a state in which it shares binding
between the two arginines in the WT protein to one in which it binds
exclusively to only one in the mutants. Consistent with a shared
contribution of Arg106 and Arg126 to the
binding affinity, the affinity of a double R106Q/R126Q mutant in A-FABP
is about 240-fold lower than the WT, roughly consistent with the sum of
the reductions (160-fold) for the single mutants (data not shown).
Finally, these results predict that in A-FABP the locations of the FA
carboxylate should differ considerably in R106A and R126A and would be
more ordered, while, as discussed previously (16), in I-FABP the
location for R106A would not differ appreciably from WT but would
exhibit less order.
Interactions along the Hydrocarbon Chain of the FA Mutations That
Have Little Effect on FA Binding Affinities--
Residues at position
40 (Met) in the adipocyte protein and position 115 in A-FABP and I-FABP
(Val and Gln, respectively), interact with the FA in the region near
the carboxylate (Refs. 12 and 13 and Fig. 1). Although these residues
are within van der Waals distances of the FA, Ala substitutions at
these positions have virtually no effect on affinities and relatively little effect on the underlying thermodynamic parameters. Residues Thr29 and Val32 in A-FABP and Lys27
in I-FABP are within about 4.5 Å of the terminal end of the FA and
have been identified as amino acid residues that are part of the portal
region of these proteins (2, 3). Nevertheless, Ala, Cys, and Phe
substitutions for Lys27 in I-FABP or Ala for
Thr29 or Val32 in A-FABP have relatively small
effects on affinities as well as the underlying enthalpy and entropy
changes. In contrast, Ory et al. (32) found that a V32D
substitution in A-FABP reduces oleate binding by about 30-fold. This
may suggest a rather special aspartate-FA acid interaction, because, in
this same study, binding of the competitive fluorescent probe ANS was
only reduced by about 2-fold.
Mutations That Alter Affinities Similarly in A-FABP and
I-FABP--
Corresponding residues Asp76,
Met20, Val25, and Phe57 in A-FABP
and Asp74, Met18, Ile23, and
Phe55 in I-FABP interact with the FA along the terminal
portion of the hydrocarbon chain, and most of these residues contribute
to the structure of the portal region of their respective proteins. Amino acid substitutions at these positions generate proteins that have
lower affinities, which, with the exception of Met20, are
mostly due to lower |
H| values for these mutants. The
reduction in affinities for M20A in A-FABP and M18A in I-FABP are
brought about by large (6-8 kcal/mol) |
H| and
smaller |T
S|, but of opposite signs for
Met20 and Met18, making these interactions
among the most difficult to understand. The reductions in affinity for
oleate binding to F57A and F57G (Fig. 2 and Table I) are consistent
with the 3-fold lower affinity for oleate observed previously for the
F57H mutant of Ory et al. (32). Binding of arachidonate to
the F57A mutant of A-FABP reveals no affinity change (Fig. 5),
presumably because the C-18-C-20 carbons curve back into the binding
cavity rather than extending out past Phe57 as do the other
FAs (12, 13, 33). However, this lack of affinity change is a
consequence of exactly compensating changes in enthalpy and entropy of
about 4 kcal/mol rather than no change in the interactions, suggesting
that the effects of the F57A substitution are propagated to other parts
of the protein.
Mutants That Have the Opposite Effects on FA-FABP Interactions in
A-FABP and I-FABP--
Alanine substitutions at four corresponding
positions in A-FABP and I-FABP generate proteins with affinities and
thermodynamic parameters that are altered differently in the adipocyte
and intestinal proteins. These results emphasize the subtle nature of
molecular interactions that govern ligand binding and suggest why
visual inspection of the structures alone will not in general predict correctly the effect of mutagenesis. 1) For C117A in A-FABP and Y117A
in I-FABP, affinities, relative to WT, are about 3-4-fold lower for
A-FABP and 5-fold greater for I-FABP. Lower affinities result because
|
H| decreases more than entropy increases in A-FABP, while in I-FABP increased affinities are the result of small, not quite
compensating changes in enthalpy and entropy (Fig. 6). 2) For Y19A in
A-FABP and F17A in I-FABP, affinities decrease about 60-fold and less
than 2-fold, respectively. The magnitude of these changes does not,
however, reflect the underlying interactions, because
|
H| are actually larger for F17A than Y19A.
However, because the entropy changes are highly compensatory in I-FABP but not in A-FABP, the affinity changes more for the adipocyte than for
the intestinal protein. 3) Phe16 in A-FABP and
Tyr14 in I-FABP reveal behavior similar to
Tyr19 and Phe17. 4) Ala substitutions for
Lys58 in A-FABP and Arg56 in I-FABP, which are
located on the surface near their respective portal regions, result in
small (50%) and 20-fold decreases in affinity for K58A and R56A,
respectively. In both proteins |
H| is reduced by 1-2
kcal/mol, but in K58A entropy increases by about the same amount and to
a much smaller degree in R56A.
Summary--
These studies reveal substantial differences in the
interaction of FA with adipocyte and intestinal FABPs. Although their amino acid sequences are the basis for this difference, how the different amino acid residue-FA interactions contribute to the binding
affinity is not apparent from a simple inspection of the two
structures, primarily because of entropy/enthalpy compensation. Thus,
as indicated in Table III, roughly equivalent amino acid residue-FA
interactions can be compared in A-FABP and I-FABP. This comparison
reveals that Ala substitutions at seven of the 12 sites have equivalent
effects on affinities in the two proteins; either they have no effect
or they reduce affinities. This similarity of effects on affinities
does not, however, arise because of similarities in the underlying
interactions. As is apparent, in Table III similar effects on
affinities can arise through different combinations of the magnitude
and signs of the enthalpy and entropy components. Further complicating
the comparison of the two proteins from their structures are the
differences in the heat capacity contributions to the interactions that
can be altered significantly by specific amino acid substitutions.
Thus, as emphasized previously (16, 22), in addition to high quality
structural information an accurate understanding of the interactions of
FA and FABPs requires a complete thermodynamic characterization of
binding.