(Received for publication, April 21, 1997, and in revised form, May 2, 1997)
From the Medical Biology Institute, La Jolla, California 92037
Site-specific variants of rat intestinal fatty acid-binding protein were constructed to identify the molecular interactions that are important for binding to fatty acids (FAs). Several variants displayed affinities that appeared incompatible with the crystal structure of the protein-FA complex. Thermodynamic measurements provided an explanation for these apparent inconsistencies and revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory. These results demonstrate that understanding the effects of amino acid replacements on ligand binding requires measurements of enthalpy and entropy, in addition to affinity.
Fatty acid-binding proteins (FABPs)1
are approximately 15-kDa cytosolic proteins that may play important
roles in fatty acid (FA) trafficking (1-3). X-ray crystallography
reveals that the FA binding site is an internal cavity in the protein
(4-8). Crystal structures of rat intestine FABP (I-FABP) and its
complex with FA show that the hydrocarbon chain of the FA interacts
directly with about 19 amino acid resides and several bound waters
within this cavity (2, 4, 9). Binding of FA to FABP involves desolvation of the FA followed by insertion into the binding cavity. The net free energy for these steps for wild type I-FABP is
approximately 10 kcal/mol (10, 11) and is predominately enthalpic
(10-12).
To understand how the amino acid residue-FA interactions revealed by
the crystal structure contribute to the energy of binding we have used
site-specific mutagenesis to alter amino acid residues within the
binding cavity. Most mutagenesis studies aimed at understanding ligand
binding interactions have relied on changes in affinity to determine
which residues play important roles in the active site (13, 14).
However, the affinity is related to the free energy change
(Kd = (e (
G0/RT))) and, through
G0 =
H0
T
S0, to the enthalpy and entropy
changes of the binding reaction.2 Because
mutations alter both enthalpic and entropic contributions to ligand
binding, the changes in the underlying molecular interactions may not
be correlated with changes in affinity (15, 16). In the present study,
therefore, we have determined the free energy, enthalpy, and entropy
changes of binding for each mutant interacting with long chain FAs.
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 (17). Mutant and WT proteins were expressed in the pET/BL21 system as described (18). Protein for all mutants except E51A and F93A were isolated from cell lysates. E51A and F93A were expressed as inclusion bodies and were solubilized by denaturation in 4 M GdnHCl followed by renaturation by dialysis against a buffer consisting of 10 mM HEPES, 150 mM NaCl, 5 mM KCl, and 1 mM NaHPO4, at pH 7.4. This buffer was also used in all the binding measurements. Protein purification and delipidation for all proteins was done as described (18). ADIFAB was prepared from acrylodan-derivatized I-FABP as described (19) and is available from Molecular Probes, Eugene, OR.
Fatty Acid Binding to FABPMeasurements 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 (10). For each combination of FA
and FABP, a binding isotherm was measured for each temperature and in
all cases showed a stoichiometry of 1 FA binding per FABP monomer. The
temperature dependence of the Kd values for each FA
and FABP exhibited linear van't Hoff behavior, and
H0 values were determined from the slopes of
each of the van't Hoff plots. For the results shown in Figs. 2 and 3,
G0 values were evaluated from the
Kd values measured at 25 °C, and
T
S0 was calculated as
G0
H0.
To obtain information about the nature of the FA-FABP interaction
we constructed 24 mutants of I-FABP and measured their binding to FA.
We substituted Ala for 16 of the 19 residues interacting with the FA in
the cavity (Fig. 1A), as well as Gln at
residue 106. We also investigated Ala substitutions of the more distal amino acid residues shown in Fig. 1B. FA binding to each
mutant was measured as a function of temperature using the fluorescent probe ADIFAB (10, 19). Dissociation constants (Kd) determined at 37 °C for binding of 6 long chain FA to the wild type
protein and to each of the 24 mutants were found to range between 0.5 and 4500 nM (Table I and Fig. 3). These
results show that substitution of single residues in I-FABP can result
in proteins that bind with affinities ranging from 30-fold higher
(Leu-72, Arg-106) to almost 30-fold lower (Met-18, Phe-68) than the
wild type (WT) protein.
|
From the temperature dependence of the Kd values we
determined the differences in enthalpy
(H0), entropy
(T
S0), and free energy
(
G0) of FA binding between each mutant and
the WT protein. These results, arranged so that the
G0 values for linoleate increase
monotonically, are shown in Fig. 2 and demonstrate that
changes in affinity, equivalent to
G0
values, are not correlated with
H0 and
T
S0. This lack of correlation
results because
H0 and
T
S0 tend to compensate in these
binding reactions. As a consequence, the mutation-induced changes in
binding enthalpy and entropy are almost always larger than
G0, so that relatively small mismatches in
the enthalpy/entropy compensation can result in highly significant
changes in binding affinity. A striking feature of these results is
that the changes in affinity are not related uniquely to the changes in
enthalpy and entropy. For example, although Ala substitutions for
Leu-72, Arg-106, and Tyr-117 all result in substantial increases in
affinity, the molecular interactions that generate these increases are
different in each case. The increase in affinity for L72A is caused by
an increase in |
H0| with a smaller
decrease in entropy, that for R106A is caused by a decrease in
|
H0| with a larger increase in entropy,
and that for Y117A by both of these kind of changes, but generally with
smaller magnitudes. At the other end of the scale, M18A and F68A have
substantially lower affinities than the WT protein. This is achieved by
quite large (>6 kcal/mol) decreases in
|
H0| with smaller increases in entropy
for M18A but quite modest (<2 kcal/mol) changes in entropy and
enthalpy, that are not quite compensatory, for Phe-68, which has the
smallest affinity of any mutant. In the cases of Tyr-14, Leu-78, and
Asn-11, Ala substitutions result in virtually no change in affinity and
the appearance of no interaction between the FA and the WT residue.
However, for Y14A and L78A, the lack of change in affinity results from
quite large but virtually exactly compensating changes in enthalpy and entropy, although of opposite signs for the two sites. Only for N11A is
the lack of a change in affinity consistent with virtually no change in
the underlying molecular interactions.
An especially clear example of the value of measuring binding enthalpy
and entropy is provided by the results for Arg-106. The crystal
structure of I-FABP suggests that electrostatic interactions between
the carboxylate oxygens of the FA and the NH groups of Arg-106
contributes significantly to FA binding (2, 4, 12). Surprisingly,
elimination of these interactions by the R106A substitution results in
an up to 28-fold increase in binding affinity (Table I and
Fig. 3). Also shown in Fig. 3 are the results of a Gln substitution for Arg-106. This mutant, for which Gln-106 would be
expected to have a reduced level of attractive electrostatic interactions relative to Arg-106, reveals at most a modest reduction in
FA binding, with the exception of palmitate. Examination of Fig. 3,
however, reveals that although G0 is
decreased, increased, or unchanged by the Ala and Gln mutations, both
these mutations result in large reductions (average of about 5 kcal/mol) in the enthalpy of binding. At the same time, an increase in
entropy either compensates for (R106Q) or is significantly greater than
(R106A) the loss in enthalpy. These results illustrate that using
site-directed mutagenesis to assess the role of individual residues in
ligand binding requires an accurate understanding of how the
H0 and
T
S0 values are generated at the
structural level.
These results indicating significant increases in binding entropy for the Arg-106 mutants are consistent with the crystal structures of the WT and R106Q mutant complexed with oleate (9). These structures reveal that for the C-5 to C-18 portion of the FA hydrocarbon chain, the conformation and degree of order are similar in the WT and R106Q mutant. However, C-4 to C-1 of oleate exhibits considerably greater disorder in R106Q than in the WT protein. This suggests that the loss of an attractive electrostatic interaction in the mutant, as reflected by the decrease in enthalpy of binding, is counteracted by an increase in entropy derived, at least in part, from the increase in disorder of C-4-C-1 of the FA.
The somewhat ambiguous role of Arg-106 in FA binding raises the
question of what other interactions are responsible for binding within
the cavity. Analysis of the crystal structure suggests that FA binding
is due to a "series of feeble forces" resulting from protein-FA
interactions along the length of the FA (2, 9). Our results (Fig. 2 and
Table II) are consistent with this notion because in all
cases |G0|
2 kcal/mol. At the same
time, however, the variation of
H0 and
T
S0 for these mutants suggest
that at the molecular level a wide spectrum of interactions contribute
to FA binding, with many being quite strong
(
H0 > 5 kcal/mol). Several of the mutants
illustrate that large enthalpy changes of either sign can occur even in
the absence of apparent electrostatic interactions. For instance, an
Ala substitution of Leu-102 results in an average 4.7 kcal/mol more
favorable enthalpy while the enthalpy of the M18A mutant has a less
favorable enthalpy by an average of about 6.5 kcal/mol. In contrast to
both of these examples in which large changes in the underlying
molecular interactions are reflected in large changes in binding, the
Y14A mutant reveals almost no change in binding, but up to 6 kcal/mol
of almost exactly compensating
H0 and
T
S0.
|
If the thermodynamic parameter differences for each Ala substitution
indicate the individual interaction energies between the FA and the
amino acid residues for the WT protein, then the sum of the individual
interactions should be comparable with the total binding energies for
the WT protein. The energies of Fig. 2 and Table II reflect
interactions only within the cavity, while the measured
G0 values for binding include interactions
involved in the desolvation step. A significant portion of the
desolvation free energy is entropic (10), and the last row of Table II
shows the thermodynamic parameters with this contribution removed. As
Table II indicates, the sums of the thermodynamic contributions for all
of the mutants are significantly different from the measured
G0,
H0, and
T
S0 values. That these sums do not
equal the actual binding energies is not surprising because the
G0,
H0, and
T
S0 values are generally not
independent (16) and the values for the mutants may not accurately
reflect the interactions in the WT protein (15). Studies of the gene V
protein of bacteriophage f1 suggest that additivity might be more
accurate for well separated mutations (20). An improvement is obtained
by restricting the summation for the I-FABP mutants to 7 well-separated
residues out of the total 15 cavity residues (the asterisk row of Table II), but significant differences remain. In particular, the sums of the
cavity enthalpies differ systematically with double bond number,
although the measured binding enthalpy is virtually identical (
11
kcal/mol) for each of the 4 FA (Table II). This variation may reflect
interactions internal to the FA that differ in the bound and free
state, and this difference may be different for each of the FA (21) and
would be consistent with a compensating entropy as discussed previously
(10). Thus accurate estimates of the FA's internal energy differences
in the solvent and I-FABP bound states would help to determine whether
summation over mutation-induced changes in thermodynamic parameters
provides an accurate estimate of the total binding energetics within
the cavity.
Some of the substituted I-FABPs involved residues that are more distal
to the FA binding site (Fig. 1B). Perhaps the most interesting of these involves Arg-126, which appears to play a direct
role in FA binding to all FABPs except intestine where it is more than
6.5 Å from the FA (3). Nevertheless, as Fig. 2 shows, R126A has a
substantial effect on binding due to large H0 and
T
S0. Because very similar
changes are produced by the D34A mutation (Fig. 2), and because Arg-126
forms an electrostatic interaction with Asp-34 (2, 3, 9), the large
effect on FA binding by both of these mutations suggests that this
electrostatic bond is important, perhaps for the conformation of the
binding cavity. Relatively small
H0 and
T
S0 at distal locations can
also profoundly affect ligand binding as illustrated by Ala mutations
of Phe-47 and Phe-68, which together with Phe-62 form a barrier between
the interior and exterior of the protein and are
4.5 Å from the FA
at the carboxylate end (2). These mutations result in substantial
reductions in affinity and indeed the F68A mutation produced the
largest
G0 (~1.9 kcal/mol) of all 24 mutants. These alterations of
G0 are,
however, achieved by relatively small (generally less than 2 kcal/mol)
non-compensating changes in
H0 and
T
S0 (F68A is the only mutant not
exhibiting enthalpy/entropy compensation). The quite small effect for
the relatively neutral residue Asn-11, which is predicted to be
involved in the initial binding step (2) but is >10 Å from the FA,
demonstrates that not all residues affect FA binding.
The ability of FABPs to discriminate among different FA is an important issue in FA metabolism. In general FA metabolism exhibits a high degree of FA specificity, distinguishing clearly among FA on the basis of chain length and saturation (for example, Ref. 22). Presumably, FA recognition by proteins occurs at various steps in FA metabolism. Although FA binding to FABPs does not reveal the kind of selectivity observed in cellular metabolism (18, 19), FABPs might provide a good model of how selectivity can be built into FA recognition. Unfortunately, most of the mutants generated little change in binding specificity, and much of the specificity apparent in Table I (for example, Kd values for all mutants are in the order SA<OA<LA<LNA) is a reflection of FA solubility differences (10, 18, 19). However, a few of the mutants such as R106A, W82A, Y117A, Y14A, F93A, and M21A have significant effects on binding specificity. In the case of Trp-82, for example, the Ala mutation results in a monotonic increase in binding with double bond number for the 18-carbon series of FA, producing a 10-fold increase in binding of linolenate (18:3) relative to stearate (18:0). The molecular details of how these alterations are achieved are unclear, although the predominance of aromatic residues within the I-FABP binding cavity and the frequent deviations of arachidonate's (20:4) thermodynamic parameters relative to the other FA raise the possibility that the aromatic double bond interaction may play a role in modulating FA binding specificity.