Thermodynamics of Fatty Acid Binding to Engineered Mutants of the Adipocyte and Intestinal Fatty Acid-binding Proteins*

Gary V. RichieriDagger §, Pamela J. LowDagger , Ronald T. OgataDagger , and Alan M. KleinfeldDagger

From the Dagger  Medical Biology Institute and § Lidak Pharmaceuticals Inc., La Jolla, California 92037

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (Delta Cp) upon FA binding, and substitutions at residues Arg106 and Arg126 reduce the magnitude of Delta Cp.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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.

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 Delta H0 values were determined from the slopes of each of the van't Hoff plots.2 Delta G0 values were evaluated from the Kd values measured at 25 °C, and -TDelta S0 was calculated as Delta G0 - Delta 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 Delta H0 and -TDelta S0 as well as for Delta 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 (lambda ext = 290 nm; lambda 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 Delta G, from Delta 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 Delta G with [GdnHCl], and the free energy difference (Delta Gapp) between the native and denatured states extrapolated to zero [GdnHCl].

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (Delta Delta 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 (Delta Delta G0 = +2 kcal/mol) to 30-fold higher (Delta Delta 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 Delta Delta G0 = Delta G0 (mutant) - Delta 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 Delta Delta G0 = Delta G0 (mutant) - Delta G0 (WT) in kcal/mol, were determined at 37 °C for FA binding to the intestinal proteins.

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 (Delta 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 Delta Cp = 620 kcal/mol/K and Delta Cp = 0 kcal/mol/K for the V115A of A-FABP and the Q115A of I-FABP mutants, respectively.

Results at 25 °C for the differences in enthalpy (Delta Delta H0), entropy (TDelta Delta S0), free energy (Delta Delta G0), and heat capacity (Delta Delta Cp0) of oleate and arachidonate binding between each mutant and the WT adipocyte protein have been arranged in Fig. 5 so that the Delta Delta G0 values increase monotonically. The results demonstrate that changes in affinities (Delta Delta G0) are not correlated with Delta Delta H0, TDelta Delta S0, or Delta Delta Cp0 because |Delta Delta H0| and |TDelta Delta S0| are large, relative to |Delta Delta 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 Delta Delta 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 |Delta Delta H0| and |TDelta Delta 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 |Delta Delta 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 Delta Delta G0, Delta Delta H0, TDelta Delta S0, and Delta Delta Cp0 were calculated as Delta Gmut0 - Delta GWT0, Delta Hmut0 - Delta HWT0, TDelta Smut0 - TDelta SWT0, and Delta Cp mut0 - Delta Cp WT0, respectively. Delta G0, Delta H0, TDelta S0, and Delta Cp0 (Delta 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 Delta Delta G0 values for oleate increase monotonically. The scale for Delta Delta G0 is amplified 3-4-fold relative to those for Delta Delta H0, and TDelta Delta S0. Negative values for Delta Delta H0 and -TDelta Delta S0 indicate more favorable binding. S.D. values (in kcal/mol) are approximately 0.1-0.2 for Delta Delta G0 and 1-2 for Delta Delta Ho (indicated) and TDelta Delta S0, and values are indicated for Delta Delta Cp0. *, not done.


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Fig. 6.   Mutant-WT changes in thermodynamic parameters for I-FABP proteins. Values for Delta Delta G0, Delta Delta H0, and TDelta Delta S0 were calculated as in Fig. 5. Delta G0, Delta H0, and -TDelta 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 Delta Delta G0 values for linoleate increase monotonically. The scale for Delta Delta G0 is amplified 2-3-fold relative to those for Delta Delta H0 and TDelta Delta S0. Negative values for all three parameters indicate more favorable binding. S.D. values (in kcal/mol) are approximately 0.1-0.2 for Delta Delta G0 and 1-2 for Delta Delta H0 and TDelta Delta S0.

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 Delta Delta 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 (lambda ext = 290 nm, lambda 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 Delta Delta G denaturation for the A-FABP and I-FABP proteins are from Table II, and the Delta Delta G0 values are data from Figs. 2 and 3. The linear fit through this data has a correlation coefficient (r2) of 0.0001.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 Delta 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.

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 approx  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 (Delta Gbinding0) for the WT adipocyte and intestinal FABPs are from this study, and the Delta 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 Delta Gbinding0, Delta Gw-memb pair are indicated and abbreviated as in Fig. 2.

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, |Delta Cp| is even larger for liver than adipocyte FABP, and moreover, non-zero Delta Cp values were only observed for binding to one (high affinity) of the two liver FABP binding sites (19). Thus, Delta Cp decreases must reflect specific alterations within the adipocyte FABP binding site. Decreases in Delta 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 Delta Cp approx  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 Delta Cp. To within the fairly large uncertainties in estimating Delta Cp, the results of Fig. 5 indicate that although most mutations have relatively little affect on Delta Cp, both A and Q mutations at Arg106 and Arg126 reduce the magnitude of Delta 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 |Delta 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 Delta H and a smaller increase in entropy for R106Q but a 6 kcal/mol more favorable Delta 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 |Delta H| (3-4 kcal/mol) and almost no compensating entropy change for R126Q, to small (0-2 kcal/mol) |Delta 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 |Delta 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) |Delta Delta H| and smaller |TDelta Delta 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 |Delta 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 |Delta Delta 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 |Delta 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.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM46931.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 and reprint requests should be addressed: Medical Biology Institute, 11077 N. Torrey Pines Rd., La Jolla, CA 92037.

1 The abbreviations used are: FABP, fatty acid-binding protein; AA, arachidonate (20:4); A-FABP, adipocyte FABP; I-FABP, intestinal FABP; FA, fatty acid; LA, linoleate (18:2); LNA, linolenate (18:3); OA, oleate (18:1); PA, palmitate (16:0); SA, stearate; WT, wild type; GdnHCl, guanidine HCl.

2 Although the binding constants are given as dissociation constants (Kd), the thermodynamic parameters throughout the paper are for the association reaction, as in our previous studies (15, 19). The parameters for association are simply the negative of those for dissociation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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