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Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with
Zixi Wang1, Yaling Wu, Lanfen Li
1State Key Laboratory of Protein and Plant Gene Research, and Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing 100871, China.
Cell Research
|December 12, 2012
Summary
The circadian clock
Area of Science:
- Molecular Biology
- Structural Biology
- Chronobiology
Background:
- Mammalian circadian rhythms are regulated by core loop transcription factors CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1).
- Understanding CLOCK-BMAL1 protein-DNA interactions is crucial for deciphering circadian gene regulation.
- Previous structural studies focused on heterodimer formation, leaving DNA recognition mechanisms unclear.
Purpose of the Study:
- To elucidate the structural basis of human CLOCK-BMAL1 complex binding to E-box DNA.
- To identify novel non-canonical E-box sequences recognized by CLOCK-BMAL1.
- To investigate the role of phosphorylation in regulating CLOCK-BMAL1 DNA binding and circadian function.
Main Methods:
- X-ray crystallography to determine the structure of human CLOCK-BMAL1 bHLH domains bound to DNA.
- Systematic mutagenesis of E-box sequences to assess DNA binding affinities.
- Cell-based assays to evaluate the impact of phosphorylation on DNA binding and circadian oscillation.
Main Results:
- The crystal structure reveals hydrogen-bonding networks mediating CLOCK-BMAL1 E-box recognition.
- A hydrophobic contact indicates CLOCK-BMAL1 recognizes a 7-bp DNA element, extending beyond the canonical 6-bp E-box.
- Two high-affinity non-canonical E-boxes (AACGTGA and CATGTGA) were identified, requiring a specific flanking base pair.
- Phosphorylation mimicry at BMAL1 Ser78 inhibits DNA binding and disrupts cellular circadian rhythms.
- BMAL1 Ser78 is proposed as a key residue for integrating external signals to entrain the circadian clock.
Conclusions:
- The study provides atomic-level insights into CLOCK-BMAL1-DNA recognition, expanding the known repertoire of functional E-boxes.
- BMAL1 phosphorylation at Ser78 acts as a critical regulatory switch for circadian clock entrainment.
- These findings facilitate the identification of novel clock-controlled genes and regulatory mechanisms within the circadian system.
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