(Received 13 ApriV22 May 1995) -EJB 95 0599/3 FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non-DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-terminal domain having a great similarity to human FKBP12 (1 2-kDa FK506-binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N-labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence-specific assignment of the 'H and 15N resonances was achieved using two-dimensional and three-dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from CaH chemical shift distribution along the sequence, the short-range and medium-range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six P-strands and a three-turn a-helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The j-sheet structure, derived from long-range connectivities between backbone protons, consists of six P-strands defined as follows: B1, V22-124; B2, V32-K37; B3, D50-L61; B4, T64-S68 and F76-L80; B5, ElOO-Kl07; B6, L127-F137. They are organized in an antiparallel Psheet with the connecting topology +I, +3, +1, -3, +l. The a-helix connects strand B4 to strand B5. Globally, the structure of FKBP59-I, derived from the present work, is similar to the NMR-derived structures of uncomplexed FKBPl2. However, several conformational differences were noted at this level of structural analysis. The /3-sheet of the FKBP59 domain has an additional strand at the N-terminal and the a-helix is longer by about one helical turn. In addition, strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X-ray or NMR structures of complexed FKBP12 but not in the NMR-derived, uncomplexed structure.