SOLUTION STRUCTURE OF CYCLOSPORINE-A AND A NONIMMUNOSUPPRESSIVE ANALOG BOUND TO FULLY DEUTERATED CYCLOPHILIN

A simple strategy involving H-1 nuclear magnetic resonance (NMR) spectroscopy and complete protein deuteration was used to determine the structures of two receptor-bound drugs. A potent immunosuppressive, cyclosporin A (CsA) binds tightly to the ubiquitous and highly conserved 17.7-kDa immunophilin, cyclophilin (CyP). Fully deuterated CyP was produced by overexpressing the human CyP gene in Escherichia coli grown on deuterated algal hydrolysate in 98% D2O. As only the CsA molecule is protonated in the CsA-CyP complex, we were able to make a complete sequential assignment of the bound drug using standard two-dimensional proton NMR experiments. The structure determination was accomplished using dynamical simulated annealing calculations with a total of 124 NMR-derived distance and torsion angle restraints. Aside from binding CsA, CyP also acts as a peptidyl-prolyl cis-trans isomerase. Thus, much importance had been ascribed to the cis peptide bond present in the structures reported for free CsA in organic solvents and in crystal studies. Interestingly, CyP-bound CsA exists in an all-trans conformation with no detectable elements of regular secondary structure and no intramolecular hydrogen bonds. A nonactive CsA analog, MeAla6-CsA, was studied using the same CyP deuteration strategy. In addition to structural elucidation of the two bound drugs, we were able to differentiate between the bound and surface-exposed residues of the drugs and also validate our previous hypothesis that the single CyP tryptophan is located in the CsA-binding site. The total backbone rms deviation of the average structures of the two bound drugs was 0.54 angstrom with the primary structural difference arising from the single amino acid substitution which occurs on the solvent-exposed surface of the bound drugs. This supports recent studies which postulate that immunosuppressive drug activity may be mediated by this ''effector surface''. This method of complete protein deuteration greatly facilitates the conformational elucidation of receptor-bound drugs and identifying specific sites of intermolecular interactions and should also find great utility in detailed structural studies of other receptor-ligand complexes.