In Silico Docking of Forchlorfenuron (FCF) to Septins Suggests that FCF Interferes with GTP Binding
In Silico Docking of Forchlorfenuron (FCF) to Septins Suggests that FCF Interferes with GTP Binding
复制标题
DOI：
10.1371/journal.pone.0096390
复制
发表时间：
2014-05-02
期刊：
影响因子：
3.7
通讯作者：
Spiliotis, Elias T.
中科院分区：
文献类型：
--
作者：
Angelis, Dimitrios;Karasmanis, Eva Pauline;Spiliotis, Elias T.
Septins是一种GTP结合蛋白，可形成细胞分裂素样细丝，对真核生物的许多功能至关重要。小分子化合物，破坏隔蛋白丝组装是有价值的工具，解剖隔蛋白功能与高时间控制。到目前为止，氯吡脲（FCF）是唯一已知影响septin组装和功能的化合物。FCF抑制Septin组装的动力学，诱导形成扩大的稳定聚合物，但其潜在的作用机制尚不清楚。为了研究FCF如何结合和影响septins，我们进行了FCF对接到septins的所有可用晶体结构的计算机模拟。FCF与SEPT 2和SEPT 3的对接表明FCF优先与septins的核苷酸结合口袋相互作用。引人注目的是，预测FCF与参与GDP结合的残基形成氢键，模拟核苷酸结合。FCF与SEPT 2-GppNHp（一种不可水解的GTP类似物）和SEPT 7的结构对接表明，FCF可能呈现两种替代的非重叠构象，深入到核苷酸结合口袋的内侧和外侧。令人惊讶的是，预测FCF与P环步行者A基序GxxxxGKS/T和GTP特异性基序AKAD相互作用，所述基序GxxxxGKS/T结合GTP的磷酸盐，所述基序AKAD与GTP的鸟嘌呤碱基和高度保守的氨基酸（包括苏氨酸，其对于GTP水解是关键的）相互作用。因此，计算机模拟FCF表现出保守的结合机制，与参与GTP结合和水解的septin特征基序和残基相互作用。总之，我们的研究结果表明，FCF稳定septins锁定到一个构象，模仿核苷酸结合状态，防止进一步的GTP结合和水解。总的来说，这项研究提供了第一个深入了解FCF如何结合和稳定septins，并为合理设计FCF衍生物提供了蓝图，这些衍生物可以以更高的亲和力和特异性靶向septins。
Septins are GTP-binding proteins that form cytoskeleton-like filaments, which are essential for many functions in eukaryotic organisms. Small molecule compounds that disrupt septin filament assembly are valuable tools for dissecting septin functions with high temporal control. To date, forchlorfenuron (FCF) is the only compound known to affect septin assembly and functions. FCF dampens the dynamics of septin assembly inducing the formation of enlarged stable polymers, but the underlying mechanism of action is unknown. To investigate how FCF binds and affects septins, we performed in silico simulations of FCF docking to all available crystal structures of septins. Docking of FCF with SEPT2 and SEPT3 indicated that FCF interacts preferentially with the nucleotide-binding pockets of septins. Strikingly, FCF is predicted to form hydrogen bonds with residues involved in GDP-binding, mimicking nucleotide binding. FCF docking with the structure of SEPT2-GppNHp, a nonhydrolyzable GTP analog, and SEPT7 showed that FCF may assume two alternative non-overlapping conformations deeply into and on the outer side of the nucleotide-binding pocket. Surprisingly, FCF was predicted to interact with the P-loop Walker A motif GxxxxGKS/T, which binds the phosphates of GTP, and the GTP specificity motif AKAD, which interacts with the guanine base of GTP, and highly conserved amino acids including a threonine, which is critical for GTP hydrolysis. Thus, in silico FCF exhibits a conserved mechanism of binding, interacting with septin signature motifs and residues involved in GTP binding and hydrolysis. Taken together, our results suggest that FCF stabilizes septins by locking them into a conformation that mimics a nucleotide-bound state, preventing further GTP binding and hydrolysis. Overall, this study provides the first insight into how FCF may bind and stabilize septins, and offers a blueprint for the rational design of FCF derivatives that could target septins with higher affinity and specificity.