Molecular and Cellular Biochemistry 98: 81-93, 1990. (~) 1990 Kluwer Academic Publishers. Printed in the Netherlands. Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction James C. Sacchettini ~, Leonard J. Banaszak 2 and Jeffrey I. Gordon 3 1Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, New York, NY 10462, USA; 2Department of Biochemistry, University of Minnesota, Minneapolis, MN 55455, USA; 3Departments of Medicine and Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA Key words." fatty acid binding protein, prokaryotic expression vectors, x-ray crystallography Abstract A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE ÷ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a supE- strain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel B-strands organized into two orthogonally situated B-sheets. The overall conformation of the protein resembles that of a clam - hence the term [3-clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg 1°6and Gin 115.The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABP has a similar overall conformation to holo-I-FABP indicating that the [3-clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations 9f several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism. Comparison of the Ca coordinates of apo- and holo-I-FABP with those of other proteins indicates it is a member of a superfamily that currently includes (i) 10 mammalian intracellular lipid binding proteins, (ii) the photoactive yellow protein from the purple photoautotrophic bacterium Ectothiorhodospi- ra halophila and (iii) a group of extracellular lipid binding proteins from a diverse number of phyla that have a common ~ 'barrel' consisting of 8 anti-parallel [3-strands stacked in two nearly orthogonal sheets. In summary, E. coli-derived I-FABP not only represents a useful model for assessing the atomic details of fatty acid-protein interactions and the mechanisms which regulate acquisition and release of this type of ligand, but also structure/function relationships in other superfamily members. Abbreviations." I-FABP - Intestinal Fatty Acid Binding Protein, r.m.s. - root mean square