Solution structure and small angle scattering analysis of TraI (381-569).
Solution structure and small angle scattering analysis of TraI (381-569).
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DOI：
10.1002/prot.24114
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发表时间：
2012-08
期刊：
影响因子：
2.9
通讯作者：
Schildbach JF
中科院分区：
文献类型：
--
作者：
Wright NT;Raththagala M;Hemmis CW;Edwards S;Curtis JE;Krueger S;Schildbach JF
TraI是F质粒编码的切口酶，是质粒DNA从一种细菌接合转移到另一种细菌所必需的1756个氨基酸的蛋白质。虽然F TraI的N-和C-末端结构域的晶体结构已经确定，但蛋白质的中心结构域在结构上尚未探索。已知中心区域（在残基306和1520之间）通过高度进行性解旋酶活性结合单链DNA（ssDNA）和解旋DNA。在这里，我们表明，ssDNA结合位点位于残基381和858之间，我们还提出了该区域的N-末端（残基381至569）的高分辨率溶液结构。该片段折叠成被α螺旋包围的四链平行β折叠，并且类似于解旋酶如RecD和RecQ的N-末端的结构，尽管序列相似性很小。该结构支持的模型，F TraI导致从复制的RecD样结构域和随后的专业化的结构域到更多的N-末端ssDNA结合结构域和更多的C-末端结构域包含解旋酶基序。此外，我们提供的证据表明，切口酶和单链DNA结合域的TraI举行紧密的80个残基的接头序列，连接这两个域。这些结果为切口酶和ssDNA结合域之间的负协同效应提供了一个可能的物理解释。
TraI, the F plasmid-encoded nickase, is a 1756 amino acid protein essential for conjugative transfer of plasmid DNA from one bacterium to another. While crystal structures of N- and C-terminal domains of F TraI have been determined, central domains of the protein are structurally unexplored. The central region (between residues 306 and 1520) is known to both bind single-stranded DNA (ssDNA) and unwind DNA through a highly processive helicase activity. Here, we show that the ssDNA binding site is located between residues 381 and 858, and we also present the high-resolution solution structure of the N-terminus of this region (residues 381 to 569). This fragment folds into a four-strand parallel β sheet surrounded by α helices, and resembles the structure of the N-terminus of helicases such as RecD and RecQ despite little sequence similarity. The structure supports the model that F TraI resulted from duplication of a RecD-like domain and subsequent specialization of domains into the more N-terminal ssDNA binding domain and the more C-terminal domain containing helicase motifs. In addition, we provide evidence that the nickase and ssDNA binding domains of TraI are held close together by an 80-residue linker sequence that connects the two domains. These results suggest a possible physical explanation for the apparent negative cooperativity between the nickase and ssDNA binding domain.
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