| Plants will produce the defensive hormone Salicylic Acid（SA）to regulate the expression of pathogen-related（PR）genes when they encounter the invasion of pathogens.SA acts as a signal molecule to trigger the Systemic Acquired Resistance（SAR）response.In this process,as one of the receptors of SA,NPR1（Nonexpressor Of Pathogenesis-Related Genes 1）is the main regulator in the SA signaling pathway.NPR1 senses and transmits SA signals,and plays an important role in plant resistance to pathogen infection.In addition,NPR1 also plays an important role in many aspects,such as regulating the growth and development of plants,the steady state of circadian rhythm,the inherent defense of plants,and the response to abiotic stress.After sensing the SA signal,NPR1 may undergo a conformational change through the redox reaction.NPR1 undergoes a transition from oligomer to monomer to change its aggregation state.The monomeric NPR1 enters the nucleus,interacts with the downstream transcription factor TGA,and regulates the expression of PR genes.Different aggregation states of NPR1 correspond to different functions,and this change between different aggregation states is the key to the transmission of SA signals.However,it is not clear that what kind of conformational changes NPR1 will undergo after sensing SA,and which the specific mechanism of its transition from oligomerization to monomer.Therefore,in this study,we aim to resolve the structure of NPR1 to explore the regulatory mechanism of its conformational transformation through structural biology research on NPR1.In this study,we tried multiple proteins of the NPR family,obtained crystals of the full-length NPR4 protein,and resolved the structure of the oligomerization region of NPR1.Consistent with the biochemical data,NPR1 is in the state of homotetramer in the crystal structure,and its BTB/POZ domain and ANK domain are mainly involved in the homologous aggregation of NPR1 molecules.In the process of research,although the NPR4 protein has crystals,its diffraction resolution has not been improved,so the three-dimensional structure of the full-length protein has not been resolved.In subsequent experiments,through protease cleavage and secondary structure prediction experiments,we constructed a series of truncations based on conserved institutions for NPR1 and NPR4.Finally,a truncated protein with a stable dimer conformation of NPR1 was found.Through crystal optimization and collection of single-wavelength anomalous scattering data of recombinant protein seleno-derivatives,the phase problem was solved and the structure of NPR142-432 was resolved.The resolution is about 3（?）.As a protein with no known homologous structure,the structure of the NPR1oligomerization region not only provides a reference for the subsequent structure of the full-length NPR1 protein and the complex of NPR1,but also provides an important structural basis for elucidating the mechanism of NPR. |
