Expression, Purification, and Crystal Structure Determination of Recombinant Human Epidermal-Type Fatty Acid Binding Protein^,

Abstract

We describe the crystal structure of human epidermal-type fatty acid binding protein (E-FABP) that was recently found to be highly upregulated in human psoriatic keratinocytes. To characterize E-FABP with respect to ligand-binding properties and tertiary structure, we cloned the respective cDNA, overexpressed the protein in Escherichia coli and purified it to homogeneity by a combination of ion-exchange and size-exclusion chromatographic steps with a yield of 30 mg/L broth. The purified protein revealed a 5-fold higher affinity for stearic acid than for oleic and arachidonic acids. The crystal structure of recombinant human E-FABP was determined to 2.05 A and refined to an R(factor) of 20.7%. The initial residual electron density maps clearly showed the presence of a ligand, which was identified as endogenous bacterial fatty acid. Within a central cavity of 252 A(3), this ligand is bound in a U-shaped conformation, its carboxyl group interacting with tyrosine 131 and arginines 129 and 109, the latter via an ordered water molecule. The E-FABP crystal structure is unique in the FABP family because of the presence of a disulfide bridge between cysteines 120 and 127 that may be physiologically as well as pathophysiologically relevant. Cysteines 67 and 87 are also in close vicinity but in contrast do not form a disulfide bridge. We postulate that this protein belongs to a particular FABP subfamily whose members share common structural as well as functional features.

Structural Basis for Ligand Regulation of the Fatty Acid-binding Protein 5, Peroxisome Proliferator-activated Receptor β/δ (FABP5-PPARβ/δ) Signaling Pathway

“…Linoleic Acid Binds FABP5 in Two Distinct ConformationsThe FABP5-LA interactions are in general very similar to those previously described by Hohoff et al (54) in their analysis of FABP5 complexed to an E. coli fatty acid. The carboxylic headgroup of LA forms a salt bridge with Arg-129, as well as a hydrogen bond with the hydroxyl moiety of Tyr-131.…”

“…Because recombinant FABP5 co-purifies with E. coli LCFAs, delipidation/denaturation of the protein followed by refolding was performed prior to LA exposure and subsequent crystallization (42,54). The structure of apo-FABP5 was solved in the P4 3 2 1 2 space group at high resolution (1.67 Å), with the asymmetric unit comprised of a FABP5 monomer adopting the canonical iLBP fold (Fig.…”

“…The structure of apo-FABP5 was solved in the P4 3 2 1 2 space group at high resolution (1.67 Å), with the asymmetric unit comprised of a FABP5 monomer adopting the canonical iLBP fold (Fig. 1a) (54,55). Interestingly, crystals of the FABP5-LA complex grew only in the P3 2 21 space group, with the resulting 2.6-Å structure revealing four copies of protein in the asymmetric unit (Table 1 and Fig.…”

Crystal Structures of the Semireduced and Inhibitor-bound Forms of Cyclic Nucleotide Phosphodiesterase from Arabidopsis thaliana

“…Although two of these cysteines are in isolated positions, the other four are paired in close vicinity. Despite the absence of any reducing agent in the crystallization media, these authors (27) report one oxidized (Cys-120 -Cys-127) and one reduced (Cys-67-Cys-87) cystine. The remarkable feature in the case of FABP is that Cys-67 and Cys-87 are still located close enough to be able to form a covalent bond without any structural rearrangements.…”

Stabilization of Leukotriene A4 by Epithelial Fatty Acid-binding Protein in the Rat Basophilic Leukemia Cell

“…Ligand binding studies have revealed high affinity for oleate and arachidonate but little or no affinity for prostaglandin E 2 (34). Palmitic acid binding to E-FABP has been structurally defined by a combination of x-ray crystallography and high field NMR (35,36). The region of the protein in proximity to the C-5 C-6 methylene groups of the bound fatty acid is structurally restricted and excludes crystallographically ordered water.…”