Biochemical and Crystallographic Analyses of a Portal Mutant of the Adipocyte Lipid-binding Protein

Ory, Jeramia; Kane, Christopher D.; Simpson, Melanie A.; Banaszak, Leonard J.; Bernlohr, David A.

doi:10.1074/jbc.272.15.9793

Cited by 32 publications

(34 citation statements)

References 32 publications

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“…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.…”

supporting

confidence: 79%

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

Richieri¹,

Low²,

Ogata³

et al. 1998

Journal of Biological Chemistry

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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 ...

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supporting

confidence: 79%

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

Richieri¹,

Low²,

Ogata³

et al. 1998

Journal of Biological Chemistry

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“…Results of mutations of Phe57 in B-FABP and H-FABP indicate that this residue is not a major contributor to the FABP/ligand interaction [88,98]. In A-FABP, however, Phe57 appeared critical for the formation of the FA/A-FABP complex and the stability of the protein, but was not involved in determination of selectivity for ligands [101,102]. Phe57 displayed a greater mobility in A-FABP relative to H-FABP [103].…”

Section: Structure Of Fabpsmentioning

confidence: 98%

New insights into the structure and function of fatty acid-binding proteins

Zimmerman¹,

Veerkamp²

2002

Cellular and Molecular Life Sciences (CMLS)

481

360

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Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, and occur intracellularly in vertebrates and invertebrates. This review presents recent findings on the diversity of these FABPs and their proposed roles in fatty acid (FA) metabolism and other cellular processes. Special attention is paid to the structural features of the different mammalian FABP types and the physiological role of these proteins in FA transport, cell growth and differentiation, cellular signalling, gene transcription and cytoprotection. Additionally, data on FABP knockout mice and the implication of FABP in medicine are discussed.

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“…Protruding from the loop between βC and βD, F57 acts as a doorway to the so-called portal region, a proposed entrance to the binding pocket. 16,20,22 The data in Figure 3 also indicate that the ANS, troglitazone, and linoleate structures deviate from the oleate complex across the length of helix αII (G26-K37), and a part of the previous turn connecting to helix αI. This trend is most apparent in the isotropic B-factors, which represent the degree to which individual atomic electron densities are broadened as a result of spatial and dynamic disorder in the crystal (Figure 3(b)).…”

Section: Structural Changes Related To Ligand Bindingmentioning

confidence: 98%

Structural Basis for Activation of Fatty Acid-binding Protein 4

Gillilan

Ayers

Noy

2007

Journal of Molecular Biology

151

153

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Fatty acid-binding protein 4 (FABP4) delivers ligands from the cytosol to the nuclear receptor PPARgamma in the nucleus, thereby enhancing the transcriptional activity of the receptor. Notably, FABP4 binds multiple ligands with a similar affinity but its nuclear translocation is activated only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of the nuclear import of FABP4, we solved the crystal structures of the protein complexed with two compounds that induce its nuclear translocation, and compared these to the apo-protein and to FABP4 structures bound to non-activating ligands. Examination of these structures indicates that activation coincides with closure of a portal loop phenylalanine side-chain, contraction of the binding pocket, a subtle shift in a helical domain containing the nuclear localization signal of the protein, and a resultant change in oligomeric state that exposes the nuclear localization signal to the solution. Comparisons of backbone displacements induced by activating ligands with a measure of mobility derived from translation, libration, screw (TLS) refinement, and with a composite of slowest normal modes of the apo state suggest that the helical motion associated with the activation of the protein is part of the repertoire of the equilibrium motions of the apo-protein, i.e. that ligand binding does not induce the activated configuration but serves to stabilize it. Nuclear import of FABP4 can thus be understood in terms of the pre-existing equilibrium hypothesis of ligand binding.

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Biochemical and Crystallographic Analyses of a Portal Mutant of the Adipocyte Lipid-binding Protein

Cited by 32 publications

References 32 publications

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

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

New insights into the structure and function of fatty acid-binding proteins

Structural Basis for Activation of Fatty Acid-binding Protein 4

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