Tameling Wladimir I L, Vossen Jack H, Albrecht Mario, Lengauer Thomas, Berden Jan A, Haring Michel A, Cornelissen Ben J C, Takken Frank L W
Plant Pathology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1090 GB Amsterdam, The Netherlands.
Plant Physiol. 2006 Apr;140(4):1233-45. doi: 10.1104/pp.105.073510. Epub 2006 Feb 17.
Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP).
植物中的抗性（R）蛋白赋予先天免疫系统特异性。大多数R蛋白具有位于中央的NB-ARC（由凋亡蛋白酶激活因子1、R蛋白和CED-4共享的核苷酸结合衔接蛋白）结构域。对于两种番茄（Lycopersicon esculentum）R蛋白I-2和Mi-1，我们之前已表明该结构域作为一个ATP酶模块，能够在体外水解ATP。为了研究核苷酸结合和水解对I-2在植物中功能的作用，在NB-ARC结构域的保守基序中引入了特定突变。两个突变产生了自激活蛋白，这些蛋白在植物中表达时会诱导一种不依赖病原体的过敏反应。发现I-2的这些突变形式在ATP水解方面受损，但在ATP结合方面未受损，这表明I-2的ATP结合态而非ADP结合态是触发防御信号传导的活性形式。此外，在结合ADP后，该蛋白对ADP的亲和力增加，提示构象发生了变化。基于这些数据，我们提出I-2以及可能相关的R蛋白的NB-ARC结构域作为一个分子开关，其状态（开/关）取决于结合的核苷酸（ATP/ADP）。
