H-1 AND N-15 ASSIGNMENT OF NMR-SPECTRUM, SECONDARY STRUCTURE AND GLOBAL FOLDING OF THE IMMUNOPHILIN-LIKE DOMAIN OF THE 59-KDA FK506-BINDINGPROTEIN

FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligom eric complexes containing nontransformed, non-DNA binding, steroid rec eptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-term inal domain having a great similarity to human FKBP12 (12-kDa FK506-bi nding protein). FKBP59 binds immunosuppressant FK506 and has peptidylp rolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its confor mational features and to better understand its biological significance , we overexpressed and N-15-labeled this domain (149 amino acids) in E scherichia coli and initiated an NMR structural study in solution. Alm ost complete sequence-specific assignment of the H-1 and N-15 resonanc es was achieved using two-dimensional and three-dimensional homonuclea r and heteronuclear experiments. Localization of the secondary structu re elements was derived essentially from C alpha H chemical shift dist ribution along the sequence, the short-range and medium-range NOE conn ectivities and exchange kinetics of amide protons. The domain has a st ructured part comprising six beta-strands and a three-turn alpha-helix between K87 and M96. The first 17 residues are highly flexible and sh ow no regular secondary structure. The beta-sheet structure, derived f rom long-range connectivities between backbone protons, consists of si x beta-strands defined as follows: B1, V22-I24; B2, V32-K37; B3, D50-L 61; B4, T64-S68 and F76-L80; B5, E100-K107; B6, L127-F137. They are or ganized in an antiparallel beta-sheet with the connecting topology +1, +3, +1, -3, +1. The alpha-helix connects strand B4 to strand B5. Glob ally, the structure of FKBP59-I, derived from the present work, is sim ilar to the NMR-derived structures of uncomplexed FKBP12. However, sev eral conformational differences were noted at this level of structural analysis. The beta-sheet of the FKBP59 domain has an additional stran d at the N-terminal and the alpha-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, unc omplexed structure.