Escherichia coli-derived rat intestinal fatty acid binding protein with bound myristate at 1.5 Å resolution and I-FABP(Arg106→Gln) with bound oleate at 1.74 Å resolution

J. Eads, J. C. Sacchettini, A. Kromminga, J. I. Gordon

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Abstract

Rat intestinal fatty acid binding protein (I-FABP) is a 131-residue protein composed of two short α-helices (αI and αII) and 10 anti-parallel β-strands organized into two nearly orthogonal β-sheets. The structure of crystalline I-FABP with bound tetradecanoate (myristate) has been refined to a resolution of 1.5 Å and compared to the 1.2 Å structure of apo-I-FABP, the 1.9 Å structure of I-FABP:hexadecanoate (palmitate) and the 1.75 Å structure of I-FABP:9Z-octadecanoate (oleate) to determine how this model fatty acid receptor accommodates changes in the length of its fatty acid ligand. Myristate is located in the interior of the protein. A highly ordered, electrostatic network containing 7 hydrogen (H)-bonds links the OE1 and OE2 atoms of myristate's carboxylate group, the indole nitrogen of Trp82, NH1, and NH2 of Arg106, NE2, and OE1 of Gln115, and 2 interior ordered waters. The hydrocarbon chain of the bound fatty acid is slightly bent. Its convex face lies in a crevice, forming van der Waals contacts with the side chains of several hydrophobic and aromatic residues. Its concave face is exposed to an array of 8 interior ordered waters whose positions are stabilized by H-bond interactions with other waters, H-bond interactions with the side chains of polar/ionizable residues, and van der Waals contacts with the surface of the fatty acid. Addition of 2 or 4 methylenes to myristate produces remarkably little change in the positions of I-FABP's main chain and side chain atoms and interior ordered waters. The principal alterations are in the conformation of a surface opening (portal) connecting external and internal solvent and in the position of the benzene side chain of Phe55. Changes in the conformation of the portal reflect movement of two of its components: the backbone of αII and a type I turn (Ala73, Asp74) connecting two β-strands. The positions of the main chain atoms of Ala73 and Asp74 appear to be determined by their ability to form van der Waals contacts with the ω-terminus of the fatty acid. The side chain of Phe55 appears to function as an adjustable aromatic lid, located over the portal, whose position is dependent on an ability to form van der Waals contacts with a fatty acid's ω-terminus. The structure of I-FABP(Arg106→Gln) with bound oleate was refined to 1.74 Å and compared to the 1.75 Å structure of I-FABP(Arg106):oleate so that we could assess the contribution of the electrostatic network, which binds carboxylate, to the overall positioning of the fatty acid. This mutation results in destabilization of the electrostatic network as evidenced by the presence of several alternate positions for carboxylate in the electron density map. The discrete disorder of the carboxylate group is associated with movement of the hydrocarbon chain along the length of the binding pocket and extension of the methyl terminus beyond the boundary of the aromatic lid. Movement of the acyl chain does not perturb the positions of the protein's interior ordered waters or the side chain of its Phe55 residue. These findings provide insights about the potential manner in which I-FABP can accommodate fatty acids whose chain length exceeds 18 carbons. Together, our studies emphasize the contributions of 'feeble forces' (ion pair, H-bond, and van der Waals interactions) to the binding enthalpy in this model fatty acid receptor system.

Original languageEnglish
Pages (from-to)26375-26385
Number of pages11
JournalJournal of Biological Chemistry
Volume268
Issue number35
StatePublished - 1993

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