Crystallization and structure determination of a hepatitis delta virus ribozyme:: Use of the RNA-binding protein U1A as a crystallization module
Crystallization and structure determination of a hepatitis delta virus ribozyme:: Use of the RNA-binding protein U1A as a crystallization module
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DOI：
10.1006/jmbi.1999.3398
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发表时间：
2000-01-21
影响因子：
5.6
通讯作者：
Doudna, JA
中科院分区：
文献类型：
--
作者：
Ferré-D'Amaré, AR;Doudna, JA
采用一种新的结晶方法，获得了基因组型丁型肝炎病毒（HDV）核酶（一种大的球形RNA）的有序结晶。剪接体蛋白U1A的高亲和力结合位点被设计成催化RNA的一个片段，这是催化所必需的。由于蛋白质分子表面的化学变化比RNA分子表面的化学变化更大，因此蛋白质片段的存在有望促进结晶并改善晶体秩序。HDV核酶- u1a复合物易于结晶，其结构用蛋白质晶体重原子衍生化的标准技术求解。超过1200埃(2)的溶剂可及的配合物表面积涉及到晶体接触。由于蛋白质与蛋白质之间的相互作用占该埋藏区域的85%，这些晶体似乎主要是由复合物的蛋白质成分结合在一起的。我们的结晶方法应该有助于确定其他生物化学上重要的rna的结构，这些rna的蛋白质伴侣不存在或实验上难以处理。该复合物的精细模型（在20.0 ~ 2.3埃之间的所有反射中R-free = 27.9%）显示出具有深活性位点间隙的RNA。在这个空腔中没有观察到有序的金属离子晶体学。前人的生物化学结果表明它在胞嘧啶75的催化中起重要作用。该残基的嘧啶碱被埋在活性位点的底部，在一个可以提高其pK(a)值的环境中。我们认为这种高度保守的胞嘧啶可能是催化酯交换反应的一般碱。(C) 2000年学术出版社。
Well-ordered crystals of a genomic hepatitis delta virus (HDV) ribozyme, a large, globular RNA, were obtained employing a new crystallization method. A high-affinity binding site for the spliceosomal protein U1A was engineered into a segment of the catalytic RNA that is dispensable for catalysis. Because molecular surfaces of proteins are more chemically varied than those of RNA, the presence of the protein moiety was expected to facilitate crystallization and improve crystal order. The HDV ribozyme-U1A complex crystallized readily, and its structure was solved using standard techniques for heavy-atom derivatization of protein crystals. Over 1200 Angstrom(2) Of the solvent-accessible surface area of the complex are involved in crystal contacts. As protein-protein interactions comprise 85 % of this buried area, these crystals appear to be held together predominantly by the protein component of the complex. Our crystallization method should be useful for the structure determination of other biochemically important RNAs for which protein partners do not exist or are experimentally intractable. The refined model of the complex (R-free = 27.9% for all reflections between 20.0 and 2.3 Angstrom) reveals an RNA with a deep active site cleft. Well-ordered metal ions are not observed crystallographically in this cavity. Biochemical results of previous workers had suggested an important role in catalysis for cytosine 75. The pyrimidine base of this residue is buried at the bottom of the active site in an environment that could raise its pK(a) value. We propose that this highly conserved cytosine may be the general base that catalyzes the transesterification. (C) 2000 Academic Press.