NMR studies of an FK-506 analog, [U-13C]ascomycin, bound to FK-506-binding protein.

Multidimensional, heteronuclear NMR methods were used to determine the complete 1H and 13C resonance assignments for [U-13C]ascomycin bound to recombinant FKBP, including stereospecific assignment of all 22 methylene protons. The conformation of ascomycin was then determined from an analysis of NOEs observed in a 13C-edited 3D HMQC-NOESY spectrum of the [U-13C]ascomycin/FKBP. This structure is found to be quite different from the solution structure of the two forms of uncomplexed FK-506. However, it is very similar to the X-ray crystal structure of FK-506 bound to FKBP, rms deviation = 0.56 A. The methods used for resonance assignment and structure calculation are presented in detail. Furthermore, FKBP/ascomycin NOEs are reported which help define the structure of the ascomycin binding pocket. This structural information obtained in solution was compared to the recently described X-ray crystal structure of the FKBP/FK-506 complex.

High-level expression of recombinant human FK-binding protein from a fusion precursor.

The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L in Escherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a approximately 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed in E. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.