来自十字花科的II类水溶性叶绿素蛋白(WSCP)是结合叶绿素(Chl)及其衍生物的非光合蛋白。WSCP的生理功能尚不清楚,但它被认为与应激反应有关,这可能与它们的Chl结合和蛋白酶抑制(PI)活性有关。然而,必须更好地理解WSCP的双重功能和同时功能。这里,BnD22(甘蓝型油菜干旱诱导的22kDa蛋白)的生化功能,一种主要的WSCP表达在甘蓝型油菜叶中,使用重组His标记的蛋白质进行研究。我们发现BnD22是一种有效的半胱氨酸蛋白酶抑制剂,比如木瓜蛋白酶,但对丝氨酸蛋白酶没有抑制作用。BnD22能够结合Chla或Chlb以形成四聚体复合物。出乎意料的是,BnD22-Chl四聚体对半胱氨酸蛋白酶表现出更高的抑制作用,指示i)同时的Chl结合和PI活性,和ii)BnD22的PI活性的Chl依赖性激活。此外,BnD22-Chl四聚体的光稳定性在与蛋白酶结合时降低。利用三维结构建模和分子对接,我们发现Chl结合有利于BnD22和蛋白酶之间的相互作用。尽管它具有Chl结合能力,在叶绿体中未检测到BnD22,而是在内质网和液泡中检测到。此外,BnD22的C端延伸肽,在体内翻译后裂解,与亚细胞定位无关。相反,它大大提升了这个表达,溶解度,重组蛋白的稳定性。
Class II water-soluble chlorophyll proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins that bind with chlorophyll (Chl) and its derivatives. The physiological function of WSCPs is still unclear, but it is assumed to be involved in stress responses, which is likely related to their Chl-binding and protease inhibition (PI) activities. Yet, the dual function and simultaneous functionality of WSCPs must still be better understood. Here, the biochemical functions of Brassica napus drought-induced 22-kDa protein (BnD22), a major WSCP expressed in B. napus leaves, were investigated using recombinant hexahistidine-tagged protein. We showed that BnD22 inhibited cysteine proteases, such as papain, but not serine proteases. BnD22 was able to bind with Chla or Chlb to form tetrameric complexes. Unexpectedly, BnD22-Chl tetramer displays higher inhibition toward cysteine proteases, indicating (i) simultaneous Chl-binding and PI activities and (ii) Chl-dependent activation of PI activity of BnD22. Moreover, the photostability of BnD22-Chl tetramer was reduced upon binding with the protease. Using three-dimensional structural modeling and molecular docking, we revealed that Chl binding favors interaction between BnD22 and proteases. Despite its Chl-binding ability, the BnD22 was not detected in chloroplasts but rather in the endoplasmic reticulum and vacuole. In addition, the C-terminal extension peptide of BnD22, which cleaved off post-translationally in vivo, was not implicated in subcellular localization. Instead, it drastically promoted the expression, solubility and stability of the recombinant protein.