Evaluation of Isoprenoid Conformation in Solution and in the Active Site of Protein-Farnesyl Transferase Using Carbon-13 Labeling in Conjunction with Solution- and Solid-State NMR

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• 作者：Todd J. Zahn ; Markus Eilers ; Zhengmao Guo ; Mohamad B. Ksebati ; Matthew Simon ; Jeffrey D. Scholten ; Steven O. Smith ; and Richard A. Gibbs
• 刊名：Journal of the American Chemical Society
• 出版年：2000
• 出版时间：August 2, 2000
• 年：2000
• 卷：122
• 期：30
• 页码：7153 - 7164
• 全文大小：199K
• 年卷期：v.122,no.30(August 2, 2000)
• ISSN：1520-5126

文摘

The enzyme protein-farnesyl transferase (FTase) catalyzes the farnesylation of the Ras protein andother key signal transduction proteins, using farnesyl diphosphate (FPP) as the prenyl source. Inhibitors ofFTase are thus of great interest as potential novel anticancer agents. The design of such agents would beinformed by a detailed knowledge of the solution conformation of FPP, as well as its conformation in theactive site of FTase. Four bis-¹³C-labeled derivatives of farnesol and geranylgeraniol have been synthesizedand used to prepare the corresponding FPP and GGPP derivatives. The labeled farnesyl and geranylgeranylderivatives 2-7 were utilized in conjunction with solution ¹³C NMR to probe the conformation of the prenylchain in a variety of different solvents. These studies, along with molecular dynamics simulations, demonstratethat the prenyl chain exists primarily in an extended conformation. Surprisingly, this preference for the extendedconformation is solvent-insensitive; no significant change in conformation is seen with all six solventsinvestigated, including water. The [6,15-bis ¹³C]FPP analogue 8 was complexed with mammalian FTase, andthis complex was utilized in conjunction with rotational resonance MAS NMR to investigate the prenyl chainconformation when bound in the active site of this enzyme. The conformation determined from these experimentsis 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 extended conformation, both in a varietyof solvents of different polarities and in the active site of mammalian FTase.