Fluorescence Resonance Energy Transfer Analysis of Merlin Conformational Changes
Fluorescence Resonance Energy Transfer Analysis of Merlin Conformational Changes
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DOI：
10.1128/mcb.00248-09
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发表时间：
2010-01-01
影响因子：
5.3
通讯作者：
Ip, Wallace
中科院分区：
文献类型：
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作者：
Hennigan, Robert F.;Foster, Lauren A.;Ip, Wallace
2型神经纤维瘤病是一种遗传性常染色体疾病，由NF2肿瘤抑制基因双等位基因失活引起。NF2基因编码一个70 kDa的蛋白质Merlin，它是ezrinRadisin-moesin(ERM)家族的成员。ERM蛋白被认为是由其N-末端FERM结构域和C-末端尾部结构域(CTD)结合形成的封闭构象和两个结构域不相互作用的开放构象之间的转换而调节的。以前的工作表明，Merlin的肿瘤抑制功能也受到类似的调控，只有封闭形式才是活跃的。因此，了解控制其构象的机制是至关重要的。我们已经开发了一系列探针，通过荧光共振能量转移来测量Merlin构象，无论是作为纯化的蛋白质还是在活细胞中。使用这些工具，我们发现Merlin主要以单体的形式存在，以稳定的、封闭的构象存在，该构象由中心α-螺旋结构域介导。FERM-CTD相互作用对闭合构象的贡献似乎不那么重要。在磷酸化或与效应蛋白相互作用后，Merlin经历了一种微妙的构象变化，这表明了一种新的机制来调节FERM结构域和CTD之间的相互作用。
Neurofibromatosis type 2 is an inherited autosomal disorder caused by biallelic inactivation of the NF2 tumor suppressor gene. The NF2 gene encodes a 70-kDa protein, merlin, which is a member of the ezrinradixin-moesin (ERM) family. ERM proteins are believed to be regulated by a transition between a closed conformation, formed by binding of their N-terminal FERM domain and C-terminal tail domain (CTD), and an open conformation, in which the two domains do not interact. Previous work suggests that the tumor suppressor function of merlin is similarly regulated and that only the closed form is active. Therefore, understanding the mechanisms that control its conformation is crucial. We have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer, both as purified protein and in live cells. Using these tools, we find that merlin exists predominately as a monomer in a stable, closed conformation that is mediated by the central alpha-helical domain. The contribution from the FERM-CTD interaction to the closed conformation appears to be less important. Upon phosphorylation or interaction with an effector protein, merlin undergoes a subtle conformational change, suggesting a novel mechanism that modulates the interaction between the FERM domain and the CTD.