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Central Activator Keeps the Circadian Clock Ticking Print

Most living organisms have adapted their physiology and behavior to match the daily cycle of light and dark generated by the rotation of the earth, operating with a period of approximately 24 hours. Molecular machines in cells ultimately control such rhythmic behavior, the details of which—the “circadian clock”—are largely conserved. To understand the inner workings of the circadian clock, researchers from the University of Texas Southwestern Medical Center and Howard Hughes Medical Institute used ALS Beamline 8.2.1 to determine the three-dimensional structure of the transcriptional activator CLOCK:BMAL1 complex, the central positive component of the mammalian circadian clock.

Circadian Clock Disruption Can Pose a Health Risk

Nearly all species on earth have an internal clock that regulates certain biochemical processes in the body with a 24-hour, or circadian, rhythm. Disruption of a person’s normal circadian rhythm, like in the case of jet lag, insomnia, narcolepsy, sleep phase disorder, or night-shift work, has been associated with an increased occurrence of obesity, depression, diabetes, certain cancers, addiction, and other health issues. Current treatment often focuses on resetting the wake-sleep cycle using behavior therapy, bright light therapy, and chronotherapy.

The new model discovered here provides a starting point for study of how the CRY and PER proteins interact with and repress CLOCK:BMAL1. This could lead to insights into the detailed biochemical mechanisms by which this transcriptional feedback loop drives the circadian clock. A better understanding of how circadian rhythm is generated and how it can be disrupted may help to develop more effective treatments for these related diseases.

The transcription negative feedback loop of the mammalian circadian clock.

The circadian clock utilizes an operational logic involving a central negative feedback loop that turns genes on and off rhythmically with a period of roughly 24 hours. The positive component of this feedback loop is an activator complex containing two polypeptide subunits: CLOCK and BMAL1. The CLOCK:BMAL1 complex binds to specific DNA sequences called E-boxes, which are present in thousands of genes, activating gene transcription and function during the daytime. Among the genes activated by CLOCK:BMAL1 are those encoding the negative components of the circadian clock’s central feedback loop: Cryptochrome and Period. Activation of these genes leads to the accumulation of their protein products Cryptochrome (CRY) and Period (PER), which are transported from a cell’s cytoplasm to its nucleus so they can interact directly with CLOCK:BMAL1. This represses CLOCK:BMAL1-mediated transcription at night. As the amounts of the repressors CRY and PER decrease due to their autorepression and turnover, the activity of CLOCK:BMAL1 is gradually restored and a new round of transcription is activated to start a new circadian cycle. An important property of this molecular clock is that it is autonomous and maintains a roughly 24-hour periodicity even in the absence of environmental cues. This phenomenon is the end result of various components of the circadian clock interacting with each other and working together to keep the clock robust and on time.

To gain a fuller understanding of the circadian clock, researchers collected x-ray diffraction data at ALS Beamline 8.2.1 and determined the three-dimensional crystal structure of the transcriptional activator complex CLOCK:BMAL1 at 2.3-Å resolution, providing the first detailed picture of the clock’s central positive components. The subunits CLOCK and BMAL1 are unstable alone, so the natural state of CLOCK:BMAL1 is always a complex (with the two peptide chains forming a heterodimer).

Ribbon diagrams of CLOCK:BMAL1 heterodimer (center). The CLOCK (green, left) and BMAL1 (blue, right) are also shown separately to illustrate their different spatial domain arrangement. Linker regions between domains are colored in red or orange.

 

Click to watch a video of the complex in 3-D.

 

 

The peptide chains do have similar domain organization. Both contain a DNA-binding domain (called basic helix-loop-helix, bHLH) followed by two PAS (PER-ARNT-SIM) domains. However, the 3D structure of CLOCK:BMAL1 reveals that the three domains in CLOCK and BMAL1 have different spatial arrangement relative to each other. The two subunits wrap around each other tightly to form an unusual asymmetric heterodimer. While the two bHLH domains together form the DNA binding domain responsible for E-box DNA recognition and binding, the PAS domains are largely responsible for interacting with the negative components of the feedback loop (CRY and PER) and modulate the complex’s activity accordingly. The asymmetric arrangement of CLOCK and BMAL1 in the complex underscores the distinct functions of these two subunits. Specifically, the PAS-A domains from CLOCK and BMAL1 are in the middle of the structure, contributing to the tight association of the two subunits. The asymmetric arrangement of PAS-B in each subunit causes different surfaces of these domains to be exposed and to interact with different transcriptional regulators (repressors, effectors, etc.).

Model of CLOCK:BMAL1 in complex with E-box DNA. The potential interaction site with repressor CRY is indicated. The details of how CLOCK:BMAL1 activates transcription and how it interact with PER protein are not known.

Many previous studies showed that CLOCK and BMAL1 each has a different role in the complex despite having the same domain organization in their amino acid sequences. BMAL1 is a more general binding partner also found in complexes with other proteins participating in processes other than circadian rhythm, while CLOCK is specific for circadian rhythm control. The reason for such distinction was unclear before, but the asymmetric 3D structure of the CLOCK:BMAL1 complex immediately explains their functional differences.

 


 

Research conducted by: N. Huang,Y. Shan, C.A. Taylor, S.-H. Yoo, C.B. Green, and H. Zhang (University of Texas Southwestern Medical Center), and Y. Chelliah, C. Partch, and J.S. Takahashi (Howard Hughes Medical Institute, University of Texas Southwestern Medical Center).

Funding: Howard Hughes Medical Institute, American Heart Association, and the National Institutes of Health. Operation of the ALS is supported by the U.S. Department of Energy (DOE), Office of Basic Energy Sciences (BES).

Publication about this research: N. Huang, Y. Chelliah, Y. Shan, C.A. Taylor, S.-H. Yoo, C. Partch, C.B. Green, H. Zhang, and J.S. Takahashi, "Crystal Structure of the Heterodimeric CLOCK:BMAL1 Transcriptional Activator Complex," Science 337, 189 (2012).

ALS Science Highlight #259

 

ALSNews Vol. 336