Brassica Napus Drought-Induced 22-Kilodalton Protein (BnD22) Acts Simultaneously as a Cysteine Protease Inhibitor and Chlorophyll-Binding Protein.

Class II water-soluble chlorophyll proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins that bind 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 WSCPs' dual function and simultaneous functionality must still be better understood. Here, the biochemical functions of BnD22 (Brassica napus drought-induced 22-kDa protein), a major WSCP expressed in B. napus leaves, were investigated using recombinant His-tagged protein. We showed that BnD22 was a potent inhibitor of cysteine proteases, such as papain, but had no inhibitory on serine proteases. BnD22 was able to bind 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 BnD22's PI activity. Moreover, the BnD22-Chl tetramer photostability 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 the subcellular localization. Instead, it drastically promoted the expression, solubility, and stability of the recombinant protein.