Cryptochrome-Timeless structure reveals circadian clock timing mechanisms

Area of Science:

• Molecular mechanisms of circadian rhythms
• Structural biology of circadian clock proteins
• Insect physiology and adaptation

Background:

• Circadian rhythms regulate behaviors and diseases through gene expression oscillations.
• In flies, Timeless (Tim) mediates nuclear entry of Period (Per), while Cryptochrome (Cry) triggers Tim degradation in light.
• Understanding the molecular interactions within the circadian clock is crucial for deciphering its regulatory roles.

Purpose of the Study:

• To elucidate the structural basis of Cryptochrome (Cry) recognition of its target, Timeless (Tim).
• To investigate how light-sensing Cryptochrome interacts with Timeless and influences circadian clock function.
• To explore the structural mechanisms underlying Timeless polymorphism and its adaptation to different climates.

Main Methods:

• Cryogenic electron microscopy (cryo-EM) of the Cryptochrome-Timeless complex.
• Structural analysis of protein-protein interactions and cofactor binding.
• Investigating the role of protein phosphorylation in nuclear import regulation.

Main Results:

• Cry binds to a continuous core of Timeless armadillo repeats and a C-terminal Tim helix.
• Cry's flavin cofactor undergoes conformational changes, inducing large-scale rearrangements at the molecular interface.
• A phosphorylated Tim segment may regulate Importin-α binding, affecting Tim-Per nuclear import.
• The N-terminus of Tim inserts into the restructured Cry pocket, replacing the light-released C-terminal tail.

Conclusions:

• The study reveals the molecular mechanism by which light-sensing Cryptochrome recognizes and interacts with Timeless.
• Structural insights explain how Cryptochrome-mediated Timeless degradation regulates the fly circadian clock.
• The findings provide a structural basis for Timeless polymorphism, explaining adaptation to diverse climates.