Refined apoprotein structure of rat intestinal fatty acid binding protein produced in Escherichia coli

Rat intestinal fatty acid binding protein (I-FABP) is a member of a family of cytoplasmic hydrophobic ligand-binding proteins. To gain insights about the contribution of bound fatty acid to I-FABP's conformation and mechanism of ligand binding, we have determined the structure of Escherichia coli-derived rat apo-I-FABP to 1.96-Å resolution and compared it to the recently refined structure of I-FABP with bound palmitate. Both apo- and holo-I-FABP are composed primarily of anti-parallel β-strands which form two nearly orthogonal β-sheets ('β-clam'). The overall structures of the apo- and holo-I-FABP are nearly identical, with a root mean square (rms) difference of 0.37 Å between C(α) atoms, 0.38 Å between all main-chain atoms, and 0.94 Å between all side-chain atoms. However, rms differences of greater than 1.3 Å were noted for the side chains of Ile-23, Lys-27, Arg-56, Leu-72, Ala-73, and Asp-74. The space occupied by bound ligand in the core of the holoprotein is occupied in the apoprotein by ordered solvent molecules. This results in an increase in the total number of internal ordered solvent molecules from 7 in the holoprotein to 13 in apo-I-FABP. This finding, together with observed differences in the side-chain orientations of two residues (Arg-56 and Lys-27) situated over a potential opening to the cores of the apo- and holoproteins, suggests that solvent molecules play a critical role in ligand binding. Moreover, the data indicate that the β-clam structure is stable even in the absence of bound ligand.