Structural biology of proteins involved in circadian rhythms & DNA replication forks

Chronobiology has become an increasingly important research area, as the consequences of disturbed biorhythms in humans range from neurophysiological disturbances like sleeplessness and burnout to more severe conditions like metabolic syndrome, diabetes, and cancer.

Physiological processes that are regulated on a daily basis, the so-called circadian rhythms, can be found in all kingdoms of life, including humans. Synchronization of these processes to the environmental light-dark cycle requires photosensors, such as the cryptochromes in Drosophila, as well as a strictly regulated periodic inner clock network.

So far, most chronobiological studies have used behavioural, cell biological, and genetic approaches. Structural studies of clock protein functions, and explanations of how maladaptations evolve from disturbances to the periodically oscillating activities and concentrations of clock proteins, are still underrepresented in the field.

Our goal is to illuminate the structural bases of protein interactions and activities underlying these periodic processes, including a broad variety of proteins and different biological systems.

Structural and biochemical characterization of circadian clock proteins

Our methodical approaches include X-ray crystallography as well as biochemical and biophysical studies (e.g. UV/VIS, fluorescence polarization, isothermal titration calorimetry, pull-down, analytical gel filtration, native gels, CD). Atomic resolution insights into the molecular clockwork provided by our crystal structures pose new biological questions to be addressed by experiments in cell culture and living organisms.

Having solved the crystal structures of Drosophila and mouse PERIOD clock proteins as well as cryptochromes (see Figure below) followed by extensive structure-based studies, we have gained detailed insights into:

1. the phototransduction mechanism of the photosensor Drosophila Cryptochrome (dCRY), which contains FAD as a light sensing chromophore. This illustrates how light can be transduced into a structural/chemical signal within the circadian clock.

2. the protein interactions of a central clock component, i.e. murine Cryptochrome (mCRY), with PERIOD2 and the E3-ligase component FBXL3 (regulating mCRY stability) and with the CLOCK-BMAL1-E-Box complex (mediating transcriptional repression). This elucidates the network role of Cryptochrome as a central player of the mammalian circadian clock.

3. the versatile functions of the so called PAS (PER-ARNT-SIM) domains of the Drosophila and mammalian PERIOD proteins (dPER, mPER1,2,3) as interaction modules in the circadian clock. This explains, for example, how a single mutation in the Drosophila PERIOD (dPER) protein prolongs the fly’s day to about 29 hours by affecting protein interactions.

Structural and biochemical characterization of circadian clock proteins

Clock protein structures determined by our group

Recent results and future directions

Very recently, we have determined the crystal structure of the mammalian PERIOD-Cryptochrome (PER-CRY) complex (Cell, 2014). This defines the protein-protein interaction in detail and allows exact planning for future experiments.

Thus, our future goal is the investigation of interactions between various clock components. In addition, we are working on the mammalian “Timeless” protein, which (despite its significant sequence homology to the clock protein Drosophila Timeless) seems to play a minor role in the mammalian circadian clock, but has important functions in DNA replication and cell cycle control.

In future, we will continue our interdisciplinary structure-function analysis of circadian clock proteins and mammalian Timeless using purified components as well as systemic approaches. Our biological research topics as well as our methodical approaches and expertise offer broad opportunities to interact with local groups.