Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase
Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase
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DOI：
10.1021/bi0266838
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发表时间：
2003-04-08
期刊：
影响因子：
2.9
通讯作者：
Distefano, MD
中科院分区：
文献类型：
--
作者：
Turek-Etienne, TC;Strickland, CL;Distefano, MD
蛋白质法尼基转移酶 (PFTase) 催化法呢基二磷酸和蛋白质底物之间的反应，形成硫醚连接的异戊二烯化蛋白质。许多异戊二烯化蛋白质参与信号传导过程这一事实引起了人们对蛋白质异戊二烯基转移酶作为可能的抗癌靶点的极大兴趣。虽然在理解异戊二烯基转移酶如何区分相关靶蛋白方面已经取得了相当大的进展，但这些酶区分类异戊二烯的规则还不太清楚。为了阐明 PFTase 如何区分 FPP 和较大的异戊二烯二磷酸，我们结合生化和结构方法研究了该酶与几种类异戊二烯类似物 GGPP 和法尼基化肽产物之间的相互作用。两种光活性类异戊二烯类似物可抑制酵母 PFT 酶，K 值低至 45 nM。对与 PFTase 结合的这些类似物之一的晶体分析表明，当与 PFTase 结合时，二磷酸部分和两个异戊二烯单元结合在 FPP 中相应原子占据的相同位置。然而，二苯甲酮基团突出到受体蛋白结合位点并阻止第二（蛋白质）底物的结合。与 PFTase 结合的香叶基香叶基二磷酸的晶体分析表明，分子的末端两个异戊二烯单元和二磷酸基团映射到 FPP 中的相应原子；然而，第一和第二异戊二烯单元从受体蛋白结合位点凸出。 GGPP 结合模式与法磺基化肽产物的结合的比较表明，体积较大的类异戊二烯不能在不与受体蛋白产生不利的空间相互作用的情况下重排转化为产物。综合起来，这些数据并不支持“分子尺假说”。相反，我们提出了一种“第二位点排除模型”，其中 PFTase 以某种方式结合较大的类异戊二烯，从而阻止受体蛋白随后的有效结合或其转化为产物。
Protein farnesyl transferase (PFTase) catalyzes the reaction between famesyl diphosphate and a protein substrate to form a thioether-linked prenylated protein. The fact that many prenylated proteins are involved in signaling processes has generated considerable interest in protein prenyl transferases as possible anticancer targets. While considerable progress has been made in understanding how prenyl transferases distinguish between related target proteins, the rules for isoprenoid discrimination by these enzymes are less well understood. To clarify how PFTase discriminates between FPP and larger prenyl diphosphates, we have examined the interactions between the enzyme and several isoprenoid analogues, GGPP, and the farnesylated peptide product using a combination of biochemical and structural methods. Two photoactive isoprenoid analogues were shown to inhibit yeast PFTase with K, values as low as 45 nM. Crystallographic analysis of one of these analogues bound to PFTase reveals that the diphosphate moiety and the two isoprene units bind in the same positions occupied by the corresponding atoms in FPP when bound to PFTase. However, the benzophenone group protrudes into the acceptor protein binding site and prevents the binding of the second (protein) substrate. Crystallographic analysis of geranylgeranyl diphosphate bound to PFTase shows that the terminal two isoprene units and diphosphate group of the molecule map to the corresponding atoms in FPP; however, the first and second isoprene units bulge away from the acceptor protein binding site. Comparison of the GGPP binding mode with the binding of the famesylated peptide product suggests that the bulkier isoprenoid cannot rearrange to convert to product without unfavorable steric interactions with the acceptor protein. Taken together, these data do not support the "molecular ruler hypotheses". Instead, we propose a "second site exclusion model" in which PFTase binds larger isoprenoids in a fashion that prevents the subsequent productive binding of the acceptor protein or its conversion to product.