Verma Vivek, Sivaraman Jayaraman, Srivastava Anjil Kumar, Sadanandom Ari, Kumar Prakash P
Department of Biological Sciences, Faculty of Science, National University of Singapore, 117543, Singapore, Singapore.
New Phytol. 2015 Apr;206(2):726-37. doi: 10.1111/nph.13297. Epub 2015 Jan 30.
Eukaryotic two-component signaling involves the His-Asp-His-Asp multistep phosphorelay (MSP). In Arabidopsis thaliana, cytokinin-mediated MSP signaling intermediates include histidine kinases (HKs), histidine phosphotransfer proteins (Hpts) and response regulators (RRs). The structure-function relationship of interaction between Hpt (e.g. AHP1) and RR (e.g. ARR4) is poorly understood. Using a homology model and yeast two-hybrid analysis, we identified key amino acids of ARR4 at the AHP1-ΔARR4((16-175)) interaction interface. Mutating them in Arabidopsis (arr3,4,5,6,8,9 hextuple mutant background) and performing root length assays provided functional relevance, and coimmunoprecipitation (coIP) assay provided biochemical evidence for the interaction. The homology model mimics crystal structures of Hpt-RR complexes. Mutating selected interface residues of ARR4 either abolished or destabilized the interaction. D45A and Y96A mutations weakened interaction with AHP1, and exhibited weaker rescue of root elongation in the hextuple mutants. CoIP analysis using cytokinin-treated transgenic Arabidopsis seedlings provided biochemical evidence for weakened AHP1-ARR4 interaction. The relevance of the selected residues for the interaction was further validated in two independent pairs of Hpt-RR proteins from Arabidopsis and rice (Oryza sativa). Our data provide evidence of a link between Hpt-RR interaction affinity and regulation of downstream functions of RRs. This establishes a structure-function relationship for the final step of a eukaryotic MSP signal cascade.
真核生物双组分信号传导涉及组氨酸 - 天冬氨酸 - 组氨酸 - 天冬氨酸多步磷酸化中继（MSP）。在拟南芥中，细胞分裂素介导的MSP信号传导中间体包括组氨酸激酶（HKs）、组氨酸磷酸转移蛋白（Hpts）和响应调节因子（RRs）。人们对Hpt（如AHP1）与RR（如ARR4）之间相互作用的结构 - 功能关系了解甚少。通过同源模型和酵母双杂交分析，我们确定了在AHP1 - ΔARR4（（16 - 175））相互作用界面处ARR4的关键氨基酸。在拟南芥（arr3、4、5、6、8、9六重突变体背景）中对这些氨基酸进行突变并进行根长测定提供了功能相关性，免疫共沉淀（coIP）测定为这种相互作用提供了生化证据。该同源模型模拟了Hpt - RR复合物的晶体结构。对ARR4选定的界面残基进行突变要么消除了相互作用，要么使其不稳定。D45A和Y96A突变削弱了与AHP1的相互作用，并在六重突变体中表现出较弱的根伸长恢复能力。使用细胞分裂素处理的转基因拟南芥幼苗进行的coIP分析为减弱的AHP1 - ARR4相互作用提供了生化证据。在来自拟南芥和水稻（Oryza sativa）的两对独立的Hpt - RR蛋白中进一步验证了选定残基对相互作用的相关性。我们的数据提供了Hpt - RR相互作用亲和力与RR下游功能调节之间联系的证据。这为真核生物MSP信号级联的最后一步建立了结构 - 功能关系。
