Summary: A new structural study reveals how core clock proteins assemble into molecular machines that drive the circadian oscillator and sheds light on disorders linked to circadian rhythm disruption.
Source: Harvard.
A small set of molecular machines orchestrates the biochemical oscillator that times many body processes.
Circadian clocks—biochemical oscillators that regulate physiology, metabolism and behavior on an approximately 24-hour cycle—exist across animals, plants, fungi and some bacteria. While researchers have identified many of the core proteins that make up these clocks, the way these components assemble and operate as a coherent timing device has been unclear.
In a study published Sept. 7 in Molecular Cell, a team led by Harvard Medical School researcher Charles Weitz reports that key clock proteins organize themselves into a few large macromolecular assemblies. These assemblies act as molecular machines that control the precise timing of circadian rhythms and provide a structural basis for how the negative feedback loop at the heart of the clock operates.
The findings offer the first detailed structural glimpse of endogenous clock machinery. Understanding how these complexes form and function is an important step toward explaining how disruptions in the circadian system can contribute to sleep disorders, metabolic problems and cancer.
Earlier work in the late 1990s identified several critical clock proteins, including three PERIOD proteins (PER1, PER2, PER3), two cryptochrome proteins (CRY1, CRY2), and casein kinase-1 (CK1). These components accumulate in the cell, enter the nucleus and repress the activity of the CLOCK-BMAL1 transcription factor that drives production of PER and CRY. As PER and CRY levels fall, CLOCK-BMAL1 resumes activity and the cycle repeats. That feedback loop takes about 24 hours.
Until now it was widely assumed that these proteins entered the nucleus independently or in small groups to carry out distinct tasks. The Weitz laboratory’s experiments challenge that view by showing a coordinated, higher-order organization of the clock proteins.
To determine how the proteins assemble, researchers isolated protein complexes from the nuclei of mouse cells at the peak of PER/CRY-mediated repression. They discovered a single large complex that included all three PER proteins, both CRYs, and CK1, together with roughly thirty accessory proteins. Electron microscopy revealed that the nuclear complex is roughly quasi-spherical in shape and associates directly with CLOCK-BMAL1.
Although initial purification used mouse liver—an organ rich in clock proteins—the same large complex was detected in other tissues such as kidney and brain. The investigators named this universal assembly the PER complex. Its presence across tissues suggests that the six core clock proteins generally do not act alone but assemble into a coordinated repressor machine to implement the negative feedback loop.
In the cytoplasm, the researchers detected multiple precursor complexes composed of different subsets of the six core proteins. One cytoplasmic assembly contained all six proteins (referred to as the “upper complex”), while three other complexes lacked one or more components. These cytoplasmic species appear to represent stages in an assembly pathway that culminates in nuclear import of the fully formed PER complex.
Notably, the upper cytoplasmic complex included GAPVD1, a protein previously known for roles in intracellular trafficking. Although the precise role of GAPVD1 in clock function remains to be fully defined, experiments that reduced GAPVD1 disrupted normal circadian timing, supporting the idea that GAPVD1 helps regulate assembly or transport of the PER complex.
Single-particle electron microscopy provided additional structural detail. Nuclear PER complexes purified from mouse liver measured roughly 40 nm and appeared as compact, quasi-spherical structures. Purified cytoplasmic PER assemblies were smaller—around 20–25 nm—and exhibited flexibly tethered globular domains, consistent with intermediates in an assembly process that is regulated, at least in part, by GAPVD1.
Weitz emphasizes that many mechanistic questions remain. How the assembled complexes coordinate repression of CLOCK-BMAL1, how assembly is timed and regulated, and how accessory proteins influence stability and nuclear import are important open issues. Nonetheless, defining these macromolecular assemblies represents a major advance in understanding the structural organization of the mammalian circadian feedback loop.
“The circadian clock is a deep timing system that controls broad aspects of cell physiology and behavior,” Weitz said. “By revealing how these molecular machines are built, we now can begin to ask targeted questions about how they work and how their dysfunction contributes to disease.”
Co-investigators included Rajindra Aryal, Pieter Bas Kwak, Alfred G. Tamayo, Po-Lin Chiu and Thomas Walz.
Funding: Supported by the G. Harold and Leila Y. Mathers Charitable Foundation; the National Institutes of Health (R01 NS095977), NIH training grants in neurobiology and sleep/circadian research, a Mahoney postdoctoral fellowship, and the Alice and Joseph E. Brooks Fund postdoctoral fellowship.
Original research: “Macromolecular Assemblies of the Mammalian Circadian Clock” by Rajindra P. Aryal, Pieter Bas Kwak, Alfred G. Tamayo, Michael Gebert, Po-Lin Chiu, Thomas Walz, and Charles J. Weitz in Molecular Cell. Published online September 7, 2017.
Abstract
Macromolecular Assemblies of the Mammalian Circadian Clock
Highlights
• Macromolecular organization of core circadian clock proteins in cells
• Evidence for a cytoplasmic assembly pathway for PERIOD complexes prior to nuclear entry
• Electron microscopy images of nuclear and cytoplasmic PERIOD assemblies
Summary
The mammalian circadian clock is organized around a feedback loop in which PER and CRY proteins repress their own transcription. In mouse liver nuclei, all three PERs, both CRYs, and casein kinase-1δ were found together in an approximately 1.9-MDa repressor assembly that quantitatively includes its CLOCK-BMAL1 transcriptional target. Prior to this incorporation, CLOCK-BMAL1 is present in an approximately 750-kDa complex. Single-particle electron microscopy identified nuclear PER complexes as quasi-spherical ∼40-nm structures. In the cytoplasm, PERs, CRYs, and CK1δ are distributed among several complexes of ∼0.9–1.1 MDa that appear to represent an assembly pathway regulated by GAPVD1, a cytoplasmic trafficking factor. Electron microscopy of cytoplasmic complexes revealed ∼20-nm and ∼25-nm structures with flexibly tethered domains. These results define the macromolecular assemblies that compose the circadian feedback loop and provide an initial structural view of endogenous eukaryotic clock machinery.