1H and 15N Assignment of NMR Spectrum, Secondary Structure and Global Folding of the Immunophilin-Like Domain of the 59-kDa FK506-Binding Protein

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 (12-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 1H 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 CαH 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 β-strands and a three-turn α-helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The β-sheet structure, derived from long-range connectivities between backbone protons, consists of six β-strands defined as follows: B1, V22–124; B2, V32–K37; B3, D50–L61; B4, T64–S68 and F76–L80; B5, E100–K107; B6, L127–F137. They are organized in an antiparallel β-sheet with the connecting topology +1, +3, +1, -3, +1. The α-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 FKBP12. However, several conformational differences were noted at this level of structural analysis. The β-sheet of the FKBP59 domain has an additional strand at the N-terminal and the α-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.