N-15 NMR RELAXATION STUDIES OF THE FK506 BINDING-PROTEIN - DYNAMIC EFFECTS OF LIGAND-BINDING AND IMPLICATIONS FOR CALCINEURIN RECOGNITION

Backbone dynamics of the ligand- (FK506-) bound protein FKBP- 12 (107 amino acids) have been examined using N-15 relaxation data derived fro m inverse-detected two-dimensional H-1-N-15 NMR spectra. A model free formalism [Lipari & Szabo (1982) J. Am. Chem. Soc. 104, 4546-45591 was used to derive the generalized order parameter (S2), the effective co rrelation time for internal motions (tau(e)), and the chemical-exchang e line width (R(ex)) based on the measured 15N relaxation rate constan ts (R1, R2) and H-1-N-15 heteronuclear NOEs. The final optimized overa ll correlation time (tau(m)) was 9.0 ns. The average order parameter ( S2) describing the amplitude of motions on the picosecond time scale w as found to be 0.88 +/- 0.04, indicating that internal flexibility is restricted along the entire polypeptide chain. In contrast to results obtained for uncomplexed FKBP, the 80's loop (residues 82-87) surround ing the ligand binding site was found to be rigidly fixed, indicating that internal motions at this site are damped significantly due to sta bilizing noncovalent interactions with the FK506 molecule. Structural implications of these differences in picosecond mobility as well as po ssible implications for calcineurin recognition are discussed.