Abstract
Circadian timekeeping depends on protein-protein interactions as well as post-translational modifications, yet the structural details that underlie these events remain largely uncharacterized. We used AlphaFold 2.3 to predict the interaction region between the Drosophila PERIOD (PER) protein and the kinase Doubletime (DBT). The structure identified two conserved helical domains of PER, SYQ and LT, as critical for the interaction. We made mutations within these domains and used mutant period rescue constructs to assess their effects on circadian period and temperature compensation in vivo. A charge-reversal mutation (E to K) in the SYQ domain caused severe period lengthening and temperature sensitivity, which was rescued by a compensatory charge-reversal mutation in a predicted interacting LT domain residue (K to E); this validated an AlphaFold predicted interaction between two charged residues in SYQ and LT. Three additional mutations in the SYQ domain showed varying effects on period length and temperature compensation, with one uniquely displaying period shortening at higher temperatures. Most strikingly, two mutations in the LT domain (L770A, L775A) resulted in the longest recorded periods for single point mutations in period, with free-running periods > 44 hours at 25C. These also showed extreme temperature sensitivity with a magnitude of change >20 hrs. over the physiological temperatures tested. RNA-sequencing analysis of the L775A mutant shows that these behavioral changes are paralleled by altered molecular cycling. Our findings underscore the power of AlphaFold in predicting a crucial protein-protein interaction even with a largely disordered protein partner and reveal that the PER-DBT binding interface plays a crucial role in maintaining proper timekeeping and temperature-compensated circadian rhythms.
Competing Interest Statement
The authors have declared no competing interest.