The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.
The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.
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DOI：
10.1074/jbc.m115.698589
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发表时间：
2016-01-15
期刊：
影响因子：
--
通讯作者：
Cann MJ
中科院分区：
文献类型：
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作者：
Fenyk S;Dixon CH;Gittens WH;Townsend PD;Sharples GJ;Pålsson LO;Takken FL;Cann MJ
植物富含亮氨酸重复序列(NLR)的核苷酸结合蛋白使植物能够识别和响应病原菌的攻击。在此之前，我们已经证明了马铃薯的RX1 NLR能够在体外结合和弯曲DNA。DNA的原位结合需要在病原体感知后才能真正激活。然而，目前还不清楚其他NLR蛋白是否也能与DNA结合。DNA结合与免疫激活所需的NLR开关功能所固有的ATPase活性之间的关系也不清楚。在这里，我们使用与番茄I-2 NLR的N端螺旋卷曲和核苷酸结合结构域相对应的重组蛋白来研究这些问题。野生型I-2蛋白与核酸结合，偏好单链≈dsDNA和单链RNA，与RX1不同。I-2诱导DNA发生弯曲和熔融。值得注意的是，ATP增强了野生型蛋白、零P-环突变体K207R和自动激活突变体S233F中相对于ADP的DNA结合。DNA结合可激活I-2固有的ATPase活性。由于在ADP的存在下，I-2与DNA的结合减少，因此出现了一种循环机制；激活的ATP相关的I-2与DNA结合，从而促进了ATP的水解，从DNA中释放了ADP结合的I-2。因此，DNA结合是至少一部分NLR蛋白的共性，而NLR的激活与其在DNA上的活性直接相关。
Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.