| Background: The photolyase/cryptochrome protein family comprises a class of functionally different flavoproteins that share a common core of about 500 amino acids, but differ in presence/absence of N- and C-terminal extensions. Photolyases (also known as photoreactivating enzyme) are DNA repair enzymes that harvest photons of the blue spectrum to remove ultraviolet-light induced photolesions. The enzyme, its light-dependent reaction mechanism, and its 3D structure have been thoroughly characterized in various species. Cryptochromes possess a unique C-terminal extension (CT) of the core that varies in sequence and composition among species. Cryptochromes of plants and Drosophila act as circadian photoreceptor, involved in light-entrainment of the biological clock to the 24 hour rhythm dictated by solar day/night cycles. Our laboratory has performed a pioneering role in the identification and characterization of two mammalian cryptochromes. Using knockout mouse models, we have shown that mCRY1 and mCRY2 (driving rhythms with long and short periodicity, respectively) act as essential components of the circadian core oscillator that drives circadian rhythmicity. Financed by NWO-CW (program grant 700.51.304), we have extensively analyzed a set of mutant mammalian mCRY1 proteins and chimeric photolyase/mCRY1 proteins in various clock cellular assays (reporting on subcellular localization, binding protein partners, protein stability, clock function, etc). This study revealed that the C-terminus is a major determinant in shaping mCRY1 function. Apparently, the divergent evolution of the tail of mCRY1 (and likely mCRY2), distinguishing the protein from Drosophila and plant CRY proteins and hypothesized to play a role in determining circadian period length, has played an important role in establishing the mammalian clock circuit. Except for the core domain of Arabidopsis CRY proteins, information on the structure of CRY proteins is missing, which is largely due to the fact that this protein has been proven notoriously difficult (if not impossible) to overproduce and purify. Recently, we succeeded in purifying large quantities of the C-terminal extension of mCRY1 and mCRY2 (containing part of the core) and made a start with the synthesis of the biologically active chimeric photolyase/mCRY1 proteins in bacteria. Aim: The current proposal is part of a multidisciplinary effort to understand the molecular mechanism of the circadian oscillator and specifically aims at: (i) obtaining mechanistic insight in the mode of action of CRY1 and CRY2 proteins in the circadian core oscillator, with special emphasis on the function of the C-terminal extension (including identification of proteins that bind to it), (ii) determining whether photoreactivation and clock functions are mutually exclusive or whether it is possible to combine them in the same protein, (iii) determining how nature can use the same core sequence for completely different functions (e.g photoreactivation by photolyases vs clock/photoreceptor function of cryptochromes). Approach: To this end, we will purify and crystallize the CRY1 and CRY2 tails to provide structural insight in the C-terminus of mammalian CRY proteins. In addition, we intend to overexpress, purify and crystallize the current, as well as modified versions of the chimeric photolyase/CRY fusion protein to investigate if and how the core and tail domains physically interact and how this may affect structure and function. The obtained structural information will serve to design a panel of new CRY1 and CRY2 expression constructs with mutations in selected domains/structures, which will be screened for loss (or gain) of biological functions in sophisticated cellular test systems, addressing biological endpoints like subcellular localization, protein stability, transcription inhibition activity, photolyase activity, protein-protein interactions, etc. Motivation: We would like to obtain insight in the molecular mode of action of cryptochromes in the circadian oscillator and address the basic, yet highly intriguing question how cryptochromes could evolve from photolyases (i.e. which structures and domains make CRY proteins act as clock protein). Furthermore, the data generated by the fundamental work on the circadian oscillator will broaden our vision on the mechanism of the circadian clock, with implications for prediction, diagnosis and treatment of genetic circadian disorders. Partners: Whereas our laboratory has ample expertise in protein expression and purification techniques (as shown for photolyases), facilities for analysis of the 3D structure of proteins are lacking. We have found Prof.dr. T. Sixma (NKI/Erasmus MC) willing to collaborate and perform the required crystallography and X-ray diffraction studies. Once successful, we aim at inviting Dr. E. Wolf (Dortmund, Germany) to join this collaboration for co-crystallization studies with (parts of) CRY1 and PER2). |
