AtNPR4 from Arabidopsis thaliana: expression, purification, crystallization and crystallographic analysis

Salicylic acid (SA) is an important phytohormone that is involved in the regulation of plant defence, growth and development. A large number of proteins have been shown to have the ability to interact with SA, and NPR4 has been demonstrated to be a receptor of SA that plays significant roles in the innate immune response of plants. In this study, Spodoptera frugiperda (Sf9) cells were used to express full‐length AtNPR4 from Arabidopsis thaliana. To facilitate crystallization, T4 lysozyme (T4L) was added to the N‐terminus of the AtNPR4 protein. The recombinant T4L‐AtNPR4 protein was expressed, purified and crystallized using the sitting‐drop and hanging‐drop vapour‐diffusion methods. The T4L‐AtNPR4 crystals have symmetry consistent with space group C2, with unit‐cell parameters a = 93.7, b = 85.8, c = 88.2 Å, β = 90° and one molecule per asymmetric unit. The best crystal diffracted to a resolution of 2.75 Å. Structure determination is in progress. NPR4 has been shown to be a key regulator of the salicylic acid‐mediated signal perception and transduction pathways. This project aimed to obtain structural details of NPR4, thus elucidating the molecular mechanism of salicylic acid perception and its signalling role in the systemic acquired resistance pathway.