Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue
Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue
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DOI：
10.1016/j.jmb.2006.12.050
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发表时间：
2007-03-16
影响因子：
5.6
通讯作者：
Derbyshire, Victoria
中科院分区：
文献类型：
--
作者：
Van Roey, Patrick;Pereira, Brian;Derbyshire, Victoria
440 个氨基酸的 Mtu recA 内含肽由独立的蛋白质剪接结构域和核酸内切酶结构域组成。此前，去除内含肽的中心核酸内切酶结构域并进行功能选择，产生了 168 个残基的迷你内含肽 Delta I-SM，其剪接活性与全长野生型蛋白相似。 D422G 突变 (Delta I-CM) 增加了 C 末端裂解活性。使用 Delta I-SM 迷你内含肽结构（此处介绍）作为指导，我们之前通过用来自 Hedgehog 蛋白自动处理结构域的 7 个残基 U 型转弯替换残留核酸内切酶环中的 36 个氨基酸残基，生成了高活性的 139 个残基迷你内含肽，Delta Delta I-hh-SM。已确定 Delta I-SM、Delta Delta I-hh-SM 和两种变体 Delta Delta I-hh-CM 和 Delta Delta I-hh 的三维结构，以评估最小化对内含肽完整性的影响，并研究 D422G 突变的结构和功能后果。这些结构研究表明 Asp422 能够与 N 和 C 末端相互作用。 CM 变体中缺乏这些相互作用，但被与水分子的接触所取代。因此，对残基 422 进行额外的诱变，结合分离 N 端和 C 端裂解的突变，表明 Asp422 的侧链在 N 端和 C 端裂解中均发挥作用，从而表明这个高度保守的残基调节两个反应之间的平衡。 (c) 2006 Elsevier Ltd. 保留所有权利。
The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, Delta I-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (Delta I-CM) increased C-terminal cleavage activity. Using the Delta I-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, Delta Delta I-hh-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue U-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of Delta I-SM, Delta Delta I-hh-SM, and two variants, Delta Delta I-hh-CM and Delta Delta I-hh, have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions. (c) 2006 Elsevier Ltd. All rights reserved.