Functional Characterization of Conserved Positively Charged Residues Surrounding the Ligand-Binding Pocket in Plant Cryptochromes.

Plant cryptochromes are blue light-responsive photoreceptor proteins that regulate various photoresponses throughout plant growth and development. Compared with their homologous CPD I/III photolyases, plant cryptochromes have lost DNA repair activity but evolved signal transduction functions. The structurally conserved ligand-binding pocket, which corresponds to the photolyase active site, exhibits distinctive features in plant cryptochromes, with key binding residues being altered relative to photolyases. Notably, the distribution of charged amino acids around this pocket differs markedly between the two protein families. In this study, we identified two conserved positively charged residues (Arg237 and Arg357) flanking this pocket in Chlamydomonas reinhardtii cryptochrome (pCRY) and its Arabidopsis homologs. Biochemical and physiological analyses demonstrated that these residues effectively modulate photoreduction kinetics and oxidative stability of the flavin adenine dinucleotide (FAD) cofactor, participate in ATP binding, and ultimately influence flowering time in transgenic Arabidopsis plants. Our findings reveal that these conserved positively charged sites play critical roles in regulating the photosensitivity and functional stability of plant cryptochromes.