Expression, purification, and crystal structure determination of recombinant human epidermal-type fatty acid binding protein

We describe the crystal structure of human epidermal-type fatty acid bindin g protein (E-FABP) that was recently found to be highly upregulated in huma n psoriatic keratinocytes, To characterize E-FABP with respect to ligand-bi nding properties and tertiary structure, we cloned the respective cDNA, ove rexpressed the protein in Escherichia coli and purified it to homogeneity b y a combination of ion-exchange and size-exclusion chromatographic steps wi th 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 Angstrom 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 endogenou s bacterial fatty acid. Within a central cavity of 252 Angstrom(3), this li gand is bound in a U-shaped conformation, its carboxyl group interacting wi th 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 m ay be physiologically as well as pathophysiologically relevant. Cysteines 6 7 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 subfam ily whose members share common structural as well as functional features.