Evaluation of isoprenoid conformation in solution and in the active site of protein-farnesyl transferase using carbon-13 labeling in conjunction withsolution- and solid-state NMR

The enzyme protein-farnesyl transferase (FTase) catalyzes the farnesylation of the Ras protein and other key signal transduction proteins, using farne syl diphosphate (FPP) as the prenyl source. Inhibitors of FTase are thus of great interest as potential novel anticancer agents. The design of such ag ents would be informed by a detailed knowledge of the solution conformation of FPP, as well as its conformation in the active site of FTase. Four bis- C-13-labeled derivatives of farnesol and geranylgeraniol have been synthesi zed and used to prepare the corresponding FPP and GGPP derivatives. The lab eled farnesyl and geranylgeranyl derivatives 2-7 were utilized in conjuncti on with solution C-13 NMR to probe the conformation of the prenyl chain in a variety of different solvents. These studies, along with molecular dynami cs simulations, demonstrate that the prenyl chain exists primarily in an ex tended conformation. Surprisingly, this preference for the extended conform ation is solvent-insensitive; no significant change in conformation is seen with all six solvents investigated, including water. The [6,15-bis C-13]FP P analogue 8 was complexed with mammalian FTase, and this complex was utili zed in conjunction with rotational resonance MAS NMR to investigate the pre nyl chain conformation when bound in the active site of this enzyme. The co nformation determined from these experiments is in good agreement with the structure determined from crystallographic studies on the FPP-FTase complex . Thus, the isoprenyl chain of FPP exhibits a strong preference for an exte nded conformation, both in a variety of solvents of different polarities an d in the active site of mammalian FTase.