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

Abstract

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. Copyright © 1995, Wiley Blackwell. All rights reserved