Refinement of the structure of Escherichia coli-derived rat intestinal fatty acid binding protein with bound oleate to 1.75-Å resolution. Correlation with the structures of the apoprotein and the protein with bound palmitate

The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:1) has been refined with x-ray diffraction data to a resolution of 1.75 Å. The protein contains 10 anti-parallel β strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation with C1-C12 more ordered than C13-C18. Two of the eight ordered waters in I-FABP:oleate are part of a hydrogen bond network that includes the carboxylate of oleate, the guanidinium group of Arg¹⁰⁶, the nitrogen of the indole group of Trp⁸², and the side chain of Gln¹¹⁵. Most of the methylenes of bound oleate reside in a crevice formed by hydrophobic and aromatic side chains. Tyr⁷⁰ and Tyr¹¹⁷ envelop the acyl chain from C3 to C8 forming contacts with both the convex and concave faces of its van der Waals surface. The hydroxyls of each phenolic side chain hydrogen bond to ordered water molecules. Two ordered waters make van der Waals contact with the concave face of the bound fatty acid. The ω-terminal methyl of oleate is oriented so that it points toward the center of the benzene of Phe⁵⁵ allowing it to form van der Waals interactions with its component methylenes. Comparison of the structure of I- FABP:oleate with a recently refined 1.19-Å model of apoI-FABP and an earlier 2.0-Å model of I-FABP:palmitate revealed a remarkable degree of similarity in the positions of their main chain and side chain atoms and in the conformations of the bound oleate and palmitate. The principal differences were confined to a few discrete regions of the protein. The helical domain, the type I turn between β strands C and D, and the ring of Phe⁵⁵ together form a solvent-accessible portal to the interior of the protein. They are repositioned in I-FABP:oleate (and I-FABP:palmitate) so that the binding cavity is even more accessible to solvent and its volume is increased. The side chain of Phe⁵⁵ which shows discrete disorder in the apoprotein functions as an ω-terminal 'sensing device': moving progressively outward toward the surface as the chain length of the bound fatty acid increases by 2 methylenes. Tyr⁷⁰ and Tyr¹¹⁷ which also show discrete disorder in the apoprotein structure due to rotation around their C(α)-C(β) bonds, are stabilized in a single, well ordered position in the holoproteins. ApoI-FABP contains 24 waters, eight of which occupy positions virtually identical to those occupied by the eight internal waters of I-FABP oleate. The positions occupied by five of the 16 remaining waters in apoI-FABP correspond to those occupied by several methylenes of bound oleate. Together, these observations suggest that I-FABP may function as a molecular water pump, using (i) feeble forces (van der Waals contacts and hydrogen bonds) rather than prominent conformational adjustments to bind a fatty acid through interactions with ordered solvent and (ii) movement of ordered solvent to perhaps drive the binding mechanism.