New homologues of Brassicaceae water-soluble chlorophyll proteins shed light on chlorophyll binding, spectral tuning, and molecular evolution

Abstract

Type-II water-soluble chlorophyll (Chl) proteins (WSCPs) of Brassicaceae are promising models for understanding how protein sequence and structure affect Chl binding and spectral tuning in photosynthetic Chl-protein complexes. However, to date, their use has been limited by the small number of known WSCPs, which also limited understanding their physiological roles. To overcome these limitations, we performed a phylogenetic analysis to compile a more comprehensive and complete set of natural type-II WSCP homologues. The identified homologues were heterologously expressed in Escherichia coli, purified, tested for assembly with chlorophylls, and spectroscopically characterized. The analyses led to the discovery of previously unrecognized type-IIa and IIb subclass WSCPs, as well as of a new subclass that did not bind chlorophylls. Further analysis by ancestral sequence reconstruction yielded sequences of putative ancestors of the three subclasses, which were subsequently recombinantly expressed in E. coli, purified and characterized. Combining the phylogenetic and spectroscopic data with molecular structural information revealed distinct Chl-binding motifs, and identified residues critically impacting spectral tuning. The distinct Chl-binding properties of the WSCP archetypes suggest that the non-Chl-binding subclass evolved from a Chl-binding ancestor that most likely lost its Chl-binding capacity upon localization in the plant tissues with low Chl content. This dual evolutionary trajectory is consistent with WSCPs association with the Kunitz-type protease inhibitors superfamily, and indications of their inhibitory activity in response to various forms of stress in plants. These findings suggest new directions for exploring the physiological roles of WSCPs and the correlation, if any, between Chl-binding and protease inhibition functionality.

Keywords: Kunitz-type protease inhibitors; ancestral sequence reconstruction; chlorophyll-binding proteins; phylogenetic analysis; protein-ligand interactions.