The interaction of the immunophilin domain of FKBP59 (FKBP59-I) with immunosuppressant drugs was investigated by steady-state and time-resolved fluorescence of tryptophan. One of the two Trp residues present in this protein (W89), conserved in almost all immunophilins, is buried in the hydrophobic core and participates in the immunosuppressant binding. By comparison with the highly homologous protein FKBP12, containing only the buried Trp, it has been concluded that its weak fluorescence is due to an atypical H-bond interaction involving the indole nitrogen and the Phe129 benzene ring. The second Trp residue (W59) in FKBP59-I is located on the external hydrophilic side of the 50-60 beta-sheet [Craescu, C. T., Rouvière, N., Popescu, A., Cerpolini, E., Lebeau, M.-C., Baulieu, E.-E., & Mispelter, J. (1996) Biochemistry 35, 11045-11052] and is responsible for >95% of the fluorescence emission. The long lifetime of the major excited state, the large activation energy of thermal quenching, and the rotational correlation time distribution pattern suggest that its environment is not highly mobile. Binding of the immunosuppressant drugs FK506 and rapamycin leads to a approximately 60% decrease of the fluorescence intensity without any change in the fluorescence emission maximum. Time-resolved measurements show that this "quenching" is due to a conformational change which depletes the long excited-state lifetime population to the profit of a more quenched minor excited state, which becomes prominent in the complexes. This is accompanied by a strong slowing of the indole ring dynamics in the case of FK506 and by a complete immobilization in the case of rapamycin, as shown by two-dimensional (tau, theta) maximum entropy analysis of the polarized fluorescence decays. Binding of the immunosuppressant drugs therefore modifies the structure and the dynamics of the external side of the 50-60 beta-sheet in FKBP59-I, which could be relevant for the formation of ternary complexes with other protein targets.

FKBP59 is a protein usually associated with heat-shock protein hsp90 and steroid receptors. The N-terminal domain of the rabbit liver protein (149 amino acids) has a sequence homology with FKBP12, binds FK506 immunosuppressor, and has a peptidyl-prolyl cis-trans isomerase activity. The three-dimensional structure of this domain (FKBP59-I) was determined using homo- and heteronuclear multidimensional NMR spectroscopy, distance geometry, and molecular dynamics methods. Structure calculations used 1290 interproton distance restraints derived from nuclear Overhauser enhancement measurements, 29 dihedral phi angle restraints, and 92 hydrogen bond restraints. For the final 22 structures, the root mean square distance from the mean atomic coordinates, calculated for well-defined secondary structure fragments, is 0.47 +/- 0.05 and 1.26 +/- 0.15 A for backbone heavy atoms (N, C alpha, C') and for all non-hydrogen atoms, respectively. The global fold contains a twisted six-stranded antiparallel beta-sheet and a short alpha-helix packed on the hydrophobic side of the sheet. The 20 N-terminal and 12 C-terminal amino acids of the domain are disordered. The main-chain structure of FKBP59-I is globally similar to the NMR-derived and X-ray structures of unbound FKBP12. An unusual hydrogen bond interaction between the indole amino proton of Trp 89 and the aromatic cycle of Phe 129 was observed. This gives a large upfield shift (-4.8 ppm) and a significant exchange protection factor. The implications of the present structure determination on the ligand binding of FKBP59 are discussed.