Bifunctionality: New Insights into the Class of (6-4)Photolyases and animal-like Cryptochromes
The cryptochrome/photolyase family (CPF) is a huge protein family of blue-light photoreceptors, which occur in all kingdoms of life. All members of this family utilize a flavin chromophore as catalytic cofactor and show high sequence and structural similarity, although they have different functions...
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|Summary:||The cryptochrome/photolyase family (CPF) is a huge protein family of blue-light photoreceptors, which occur in all kingdoms of life. All members of this family utilize a flavin chromophore as catalytic cofactor and show high sequence and structural similarity, although they have different functions inside the living organism. Photolyases use the energy of light to repair UV-light induced DNA lesions like the cyclobutane pyrimidine dimer (CPD) or the pyrimidine-(6-4)-pyrimidone photoproduct ((6-4)PP). Cryptochromes, on the other hand, are involved in many different blue-light regulated mechanisms, like the photoperiodic flowering in plants and the entrainment of the circadian rhythm in animals.
This study focuses on the characterization of the photoreduction and (6-4) repair mechanism of the subclass of animal cryptochromes and (6-4) photolyases on the basis of the animal-like cryptochrome from the green algae Chlamydomonas reinhardtii (CraCRY). Through multiple sequence alignment with other CPFs and mutational studies, the specific residues involved in the photoreduction mechanism were successfully identified, leading to the discovery of a tyrosine as distal electron donor at the end of the conserved tryptophan triad. The photoreduction, with regard to the formation and decay of a tyrosyl radical, was extensively studied with several spectroscopic methods. All analyses resulted in the observation of an unusually long-lived tyrosyl radical upon photoreduction.
As it turned out, CraCRY is not only a cryptochrome, but also has (6-4) photolyase function, which makes it a bifunctional member of this group. To study structure-function relationships, the 3D structure of CraCRY in complex with its chromophores as well as a (6-4)PP was solved by X-ray crystallography. The structure reveals a new binding mode of the DNA lesion and provides insight into the active site. One of the essential residues for DNA repair (His1) exhibits a different conformation as in the common model, which may indicate an alternative mechanism for (6-4)PP repair.
The main knowledge about cryptochrome structures derived from the comparison with photolyases, but cryptochromes contain a highly variable C-terminal extension (CTE), which is missing in photolyases. In CraCRY this CTE is about 100 amino acids long and not shown in the solved crystal structure. For analysis of the CTE, hydrogen-deuterium-exchange coupled with mass spectrometry was used including a comparison of different reduction. Although, the coverage of the CTE was incomplete, there were significant changes between the oxidized and fully reduced state FADH− detectable. It was concluded, that the photoreduction process and the formation of the tyrosyl radical is triggering a structural change in the region between the loop carrying the tyrosyl radical and the C-terminal α22-helix.
For further investigation of the intramolecular changes upon photoreduction and DNA repair, time-resolved crystal measurements of a class II CPD photolyase (MmCPDII) and CraCRY were performed within a joint project at the free electron laser SACLA. So far, the different conformations of the flavin cofactor of MmCPDII in its different oxidation states have been successfully derived. In future, it is expected to show the whole repair mechanism for the CPD lesion as well as for the (6 4)PP by time-resolved SFX.|
|Physical Description:||244 Pages|