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

Abstract

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).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 (Ϫ11 kcal/mol) is virtually independent of FA type for I-FABP, but it decreases monotonically from Ϫ5 to Ϫ12 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 ...

Hirudin Binding Reveals Key Determinants of Thrombin Allostery

“…Furthermore, it is possible that although the listed contacts are all short range, whether van der Waals or polar, their energetic contribution to binding may be different and not necessarily proportional to their number. Finally, it is possible that the number of contacts may correlate better with the enthalpy rather than the free energy of binding, as observed in the case of intestinal fatty acid-binding protein (46). Although these alternative possibilities cannot be discounted, they likely apply to a small fraction of the large number of mutations presented in this study.…”

Fatty Acid-binding Protein-Hormone-sensitive Lipase Interaction

“…To evaluate the relationship between binding and activation, a mutant of A-FABP (R126Q) that exhibits a 100-fold lower affinity for fatty acids as measured using the ADIFAB method (25) and fluorescence binding to 1,8 ANS (Fig. 4A) was used.…”

Specificity determinants for lipids bound to β-barrel proteins

“…Finally, it should be noted that similar thermodynamic changes were observed in mutant forms of both ALBP and its intestinal homolog (12). In binding studies of many mutants of the ␤-barrel lipid binding proteins, Kleinfeld and coworkers (12) observed compensatory entropic and enthalpic changes.…”

“…In general, the binding energy is mainly derived from enthalpic effects (15). However, in single-site mutants of the protein, less favorable enthalpic contributions are often compensated for by entropic changes (12).…”

“…By way of further introduction, the structural data for many of these ␤ -barrel proteins have been supplemented with extensive studies of the affinity and the underlying thermodynamics of lipid binding (10)(11)(12). ALBP binds fatty acids in the nanomolar to low micromolar range but binds RA less tightly (13).…”