水稻白叶枯病菌Hpa1Xoo蛋白的结构分析

 在大肠杆菌中表达了水稻白叶枯病菌JxoIII菌株的Hpa1_Xoo(harpin_Xoo)蛋白,通过生物物理和生物化学方法分析了Hpa1_Xoo的结构特征。Hpa1_Xoo粗蛋白能使烟草产生过敏性坏死反应(HR),以及圆二色(CD)光谱具有α-螺旋的典型特征。通过柱层析纯化发现Hpa1_Xoo蛋白存在2种洗脱峰组分A和B,B组分的含量远高于A组分,且二组分都具有HR活性。空间排阻色谱(SEC)和分析超速离心(AUC)结构分析表明组分A是Hpa1_Xoo的二聚体形式,而组分B是Hpa1_Xoo的单体形式。组分A与B均具有α-螺旋的特征CD谱,但存在含量和构象上的差异。生物信息学方法对Hpa1_Xoo二级结构和聚体结构的预测结果与实验测定和分析拟合的结果相一致。另外,预测也发现Hpa1_Xoo存在丰富的固有无序结构区域。

The structure of Hpa1Xoo from Xanthomonas oryzae pv. oryzae

Hpa1_Xoo (harpin) is a type III secreted protein of the rice blight bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) that elicits a hypersensitive response (HR) in nonhost tobacco. The Hpa1_Xoo protein of Xoo JxoIII strain was expressed in Escherichia coli, and the structure of Hpa1_Xoo was investigated using biophysical and biochemical approaches. Hpa1_Xoo crude protein elicited HR on tobacco leaves and presented typical α-helix structure by circular dichroism (CD) spectrum compared with the empty vector pET-30a(+) crude supernatant. Hpa1_Xoo crude protein showed two peaks (A and B) indicating two components. Both fractions A and B induced HR on tobacco leaves. Fraction A was a dimer, and B was a monomer of Hpa1_Xoo analysed by size-exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) methods. Fractions A and B had the characteristics of α-helix CD spectrum, but presented differences in content and conformation. CD data fitting analysis showed that the α-helical content of fraction A was significantly higher than that of B, and fraction B contained a certain amount of β-sheet secondary structure, but A did not. The prediction results of secondary structure and oligomeric state by using programs Phyre (version 0.2), HNN, and SCORER 2.0 were consistent with the results of experimental determination and fitting analysis. Additionally, the prediction using Phyre algorithm indicated that Hpa1_Xoo protein presents five confident intrinsic disordered regions.