Discrete backbone disorder in the nuclear magnetic resonance structure of apo intestinal fatty acid-binding protein: Implications for the mechanism of ligand entry

The three-dimensional structure of the unliganded form of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) has been determined using triple-resonance three-dimensional nuclear magnetic resonance (3D NMR) methods. Sequence-specific H-1, C-13, and N-15 resonance assignments were established at pH 7.2 and 33 degrees C and used to determine the consensus H-1/C-13 chemical shift-derived secondary structure. Subsequently, an eight-stage iterative procedure was used to assign the 3D C-13- and N-15-resolved NOESY spectra, yielding a total of 3335 interproton distance restraints or 26 restraints/residue. The tertiary structures were calculated using a distance geometry/simulated annealing algorithm that employs pairwise Gaussian metrization to achieve improved sampling and convergence. The final ensemble of NMR structures exhibited a backbone conformation generally consistent with the beta-clam motif described for members of the lipid-binding protein family, However, unlike holo-I-FABP, the structure ensemble for apo-I-FABP exhibited variability in a discrete region of the backbone. This variability was evaluated by comparing the apo- and holoproteins with respect to their backbone H-1 and C-13 chemical shifts, amide H-1 exchange rates, and N-15 relaxation rates. Together, these results established that the structural variability represented backbone disorder in apo-I-FABP. The disorder was most pronounced in residues K29-L36 and N54-N57, encompassing the distal half of alpha-helix II, the linker between helix II and beta-strand B, and the reverse turn between beta-strands C and D. It was characterized by a destablization of long-range interactions between helix II and the C-D turn and a fraying of the C-terminal half of the helix. Unlike the solution-state NMR structure, the 1.2-Angstrom X-ray crystal structure of apo-I-FABP did not exhibit this backbone disorder. In solution, the disordered region may function as a dynamic portal that regulates the entry and exit of fatty acid. We hypothesize that fatty acid binding shifts the order-disorder equilibrium toward the ordered state and closes the portal by stabilizing a series of cooperative interactions resembling a helix capping box. This proposed mechanism has implications for the acquisition, release, and targeting of fatty acids by I-FABP within the cell.