Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
Wallenberg Centre for Molecular Medicine, Umeå University, Umeå, Sweden.
Life Sci Alliance. 2023 Jan 20;6(4). doi: 10.26508/lsa.202201775. Print 2023 Apr.
Type 4 secretion systems are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative type 4 secretion systems depend on relaxases to process the DNA in preparation for transport. TraI from the well-studied conjugative plasmid pKM101 is one such relaxase. Here, we report the crystal structure of the trans-esterase domain of TraI in complex with its substrate DNA, highlighting the conserved DNA-binding mechanism of conjugative relaxases. In addition, we present an apo structure of the trans-esterase domain of TraI that includes most of the flexible thumb region. This allows us for the first time to visualize the large conformational change of the thumb subdomain upon DNA binding. We also characterize the DNA binding, nicking, and religation activity of the trans-esterase domain, helicase domain, and full-length TraI. Unlike previous indications in the literature, our results reveal that the TraI trans-esterase domain from pKM101 behaves in a conserved manner with its homologs from the R388 and F plasmids.
4 型分泌系统是大型且多功能的蛋白质机器，通过水平基因转移促进抗生素耐药性和其他毒力因子的传播。可移动的 4 型分泌系统依赖于松弛酶来处理 DNA，为运输做准备。来自研究充分的可移动质粒 pKM101 的 TraI 就是这样一种松弛酶。在这里，我们报告了 TraI 的转酯化酶结构域与其底物 DNA 复合物的晶体结构，突出了可移动松弛酶的保守 DNA 结合机制。此外，我们还呈现了 TraI 的转酯化酶结构域的apo 结构，其中包含大部分灵活的拇指区域。这使我们首次能够观察到 DNA 结合时拇指亚结构域的大构象变化。我们还对 TraI 的转酯化酶结构域、解旋酶结构域和全长 TraI 的 DNA 结合、切口和重连接活性进行了表征。与文献中的先前指示不同，我们的结果表明，来自 pKM101 的 TraI 转酯化酶结构域与其来自 R388 和 F 质粒的同源物表现出保守的行为。
