Institute of Structural and Molecular Biology, UCL and Birkbeck, Malet Street, London, WC1E 7HX, UK.
Mol Microbiol. 2013 Jul;89(2):324-33. doi: 10.1111/mmi.12275. Epub 2013 Jun 14.
Relaxases are proteins responsible for the transfer of plasmid and chromosomal DNA from one bacterium to another during conjugation. They covalently react with a specific phosphodiester bond within DNA origin of transfer sequences, forming a nucleo-protein complex which is subsequently recruited for transport by a plasmid-encoded type IV secretion system. In previous work we identified the targeting translocation signals presented by the conjugative relaxase TraI of plasmid R1. Here we report the structure of TraI translocation signal TSA. In contrast to known translocation signals we show that TSA is an independent folding unit and thus forms a bona fide structural domain. This domain can be further divided into three subdomains with striking structural homology with helicase subdomains of the SF1B family. We also show that TSA is part of a larger vestigial helicase domain which has lost its helicase activity but not its single-stranded DNA binding capability. Finally, we further delineate the binding site responsible for translocation activity of TSA by targeting single residues for mutations. Overall, this study provides the first evidence that translocation signals can be part of larger structural scaffolds, overlapping with translocation-independent activities.
松弛酶是一种蛋白质，负责在接合过程中将质粒和染色体 DNA 从一种细菌转移到另一种细菌。它们与 DNA 转移起始序列中的特定磷酸二酯键共价反应，形成核蛋白复合物，随后被质粒编码的 IV 型分泌系统招募进行运输。在之前的工作中，我们鉴定了质粒 R1 上的接合松弛酶 TraI 的靶向易位信号。在这里，我们报告了 TraI 易位信号 TSA 的结构。与已知的易位信号不同，我们表明 TSA 是一个独立的折叠单元，因此形成了一个真正的结构域。该结构域可以进一步分为三个亚结构域，与 SF1B 家族的解旋酶亚结构域具有显著的结构同源性。我们还表明，TSA 是一个较大的残余解旋酶结构域的一部分，该结构域失去了解旋酶活性，但保留了单链 DNA 结合能力。最后，我们通过靶向单个残基进行突变，进一步确定了负责 TSA 易位活性的结合位点。总的来说，这项研究首次提供了证据表明易位信号可以成为更大结构支架的一部分，与非易位依赖的活性重叠。
