Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers
Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers
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DOI：
10.1016/s0960-9822(00)00621-7
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发表时间：
2000-07-27
期刊：
影响因子：
9.2
通讯作者：
Brenner, C
中科院分区：
文献类型：
--
作者：
Pace, HC;Hodawadekar, SC;Brenner, C
背景：核苷酸结合蛋白 Fhit 是肺癌中最早且最常见的失活蛋白之一，通过诱导细胞凋亡来抑制肿瘤形成。在无脊椎动物中，Fhit 被编码为与 Nit（腈水解酶超家族成员）的融合蛋白。在小鼠中，Nit1 和 Fhit 基因具有几乎相同的表达谱。根据罗塞塔石碑假说，如果可以证明单独的 Nit 和 Fhit 基因出现在同一基因组子集中（即共享系统发育谱），那么无脊椎动物中融合蛋白的存在以及小鼠中单独 mRNA 的协调表达表明 Nit 和 Fhit 在同一途径中发挥作用，并且无脊椎动物 NitFhit 的结构可能反映了 Nit-Fhit 相互作用的本质。为了确定蛋白质融合事件功能意义的系统发育谱标准，我们从具有 Fhit 同源物的生物体中克隆了其他 Nit 同源物，我们使用 ApppA 的荧光核苷酸类似物进行纯化并表征来自秀丽隐杆线虫的 NitFhit 的核苷酸特异性，结晶了 200 kDa 四聚体复合物，并通过双波长异常定相从单个汞衍生物中解析了 NitFhit 的结构结论： Nit 单体具有新的 a-P-P-a 夹心折叠，具有假定的 Cys-Glu-Lys 催化三联体。 Nit 组装成四聚体、52 链 β 盒，在相反的两极结合 Fhit 二聚体，并在复合物中部周围显示 Nit 活性位点。每个 Nit 单体的最羧基端 β 链离开 Nit 四聚体的核心并与 Fhit 相互作用，位于 NitFhit 复合物中不会改变 Fhit 二聚体的核苷酸特异性，Fhit 二聚体的方向是远离 Nit 的 ApppA 结合表面。 (C) 2000 Elsevier Science Ltd. 保留所有权利。
Background: The nucleotide-binding protein Fhit, among the earliest and most frequently inactivated proteins in lung cancer, suppresses tumor formation by inducing apoptosis. In invertebrates, Fhit is encoded as a fusion protein with Nit, a member of the nitrilase superfamily. In mice, the Nit1 and Fhit genes have nearly identical expression profiles. According to the Rosetta Stone hypothesis, if the separate Nit and Fhit genes could be shown to occur in the same subset of genomes (that is, to share a phylogenetic profile), then the existence of a fusion protein in invertebrates and the coordinated expression of separate mRNAs in mouse suggest that Nit and Fhit function in the same pathway and that the structure of invertebrate NitFhit may reflect the nature of Nit-Fhit interactions.Results: To satisfy the phylogenetic profile criterion for functional significance of protein fusion events, we cloned additional Nit homologs from organisms with Fhit homologs, We used fluorescent nucleotide analogs of ApppA to follow the purification and to characterize the nucleotide specificity of NitFhit from Caenorhabditis elegans, crystallized the 200 kDa tetrameric complex, and solved the structure of NitFhit from a single mercury derivative phased by two-wavelength anomalous diffraction.Conclusions: Nit monomers possess a new a-P-P-a sandwich fold with a presumptive Cys-Glu-Lys catalytic triad. Nit assembles into a tetrameric, 52-stranded beta box that binds Fhit dimers at opposite poles and displays Nit active sites around the middle of the complex. The most carboxy-terminal beta strand of each Nit monomer exits the core of the Nit tetramer and interacts with Fhit, Residence in the NitFhit complex does not alter the nucleotide specificity of Fhit dimers, which are oriented with ApppA-binding surfaces away from Nit. (C) 2000 Elsevier Science Ltd. All rights reserved.