Animal-Like Cryptochrome (PDB ID: 6FN3) from Chlamydomonas reinhardtii

Created by: Andrew Dudzik

The animal-like cryptochrome in Chlamydomonas reinhardtii (PDB ID: 6FN3) is a member of both the cryptochrome protein family and the photolyase protein family (1). A cryptochrome is a light-sensitive flavoprotein that generally plays a role in animal circadian rhythms and avian magnetoreceptors (2). Photolyases are also light-activated flavoproteins that contribute to DNA repair (2). Photolyases and cryptochromes are closely related except for the fact that most cryptochromes have lost the ability to repair DNA (1). Cryptochromes are separated into categories of animal type, plant type, and DASH (Drosophila, Arabidopsis, Synechocystis, and Homo) (1). What is interesting about the alga Chlamydomonas reinhardtii is that its cryptochrome is similar to those of animals despite the alga not belonging to the animal kingdom (1). Additionally, C. reinhardtii lacks the normal type of photolyase responsible for repairing UV-induced DNA lesions in the form of pyrimidine-(6-4)- pyrimidone photoadducts, or (6-4)PP (1). Such lesions form bonds between two base pairs and can impede DNA replication. One study has also shown that the animal-like cryptochrome also plays a role in genetic regulatory functions similar to photolyases (3). So based on the homology to animal cryptochromes as well as the capacity for DNA maintenance, an investigation of the structure of the cryptochrome of C. reinhardtii is important to understand how it functions in DNA lesion repair.

The primary structure of the cryptochrome consists of a single chain of 509 residues and has a molecular weight of 59895.86 D (4). While no experimental isoelectric point was recorded, the ExPASy Bioinformatics Research Portal calculated a theoretical isoelectric point of 8.34 (5). The secondary structure consists of 9 total β-sheets, 26 α-helices, and random coils (4). Approximately 6% of the sequence is contained in the β-sheets, 46% is in α-helices, and the remaining 48% of the sequence is random coils (4). Most of the residues are either hydrophobic or uncharged polar, but some acidic and basic residues are scattered within the 

α-helices and 

Two critically important residues,  His-357 and His-361, form the active site where DNA is repaired by the cryptochrome (1). This is done by forming hydrogen bonds with the covalently-linked nucleotides of the DNA lesion (1). The catalytic site formed by the two histidines is located within the middle of the molecule between the two lobes of the protein. Another critically important residue, Asn-395, controls the photochemistry of flavins bound to the photolyase through the distance of the hydrogen bond between it and the FAD molecule (1). 

The function of the animal-like cryptochrome ultimately depends on its interactions with its ligands. FAD acts as a prosthetic group for the cryptochrome due to its necessity in DNA repair and signal transduction (1). When blue light excites a tryptophan residue, specifically Trp-399, Trp-376, or Trp-322, an electron is abstracted by FAD and it is reduced to FADH with a subsequent proton transfer (1). The activated FADH then forms a hydrogen bond between the 5’ moiety of thymine in DNA and the N-6 amino group in its own adenine (1). The FADH- 5’ moiety is also stabilized by a hydrogen bond to Glu-291 while the 3’-thymine forms a hydrogen bond with two catalytically-critical histidines, His-357 and His-361 (1). Without the histidine pair, the FAD ligand would not be retained in the catalytic site, rendering the protein incapable of DNA repair. Once the DNA is in the active site, a protonated form of His-357 donates a proton to the light-activated FAD (1). Through electron and proton transfers within the DNA, a radical anion is formed on the 3’ carbon of the (6-4)PP (1). Then a proton transfers to the second critical histidine, His-361, resulting in the formation of an enol and an oxetane intermediate (1). A second photon is excited and causes another proton transfer to the oxetane intermediate, resulting in the cleavage of the C6-C4 bond in the DNA lesion (1). This cleavage of the pyrimidine-(6-4)- pyrimidone photoadducts results in repair of the lesion.

A similar structure to the animal-like Cryptochrome from C. reinhardtii is the cryptochrome of  Drosophila melanogaster (PDB ID: 4JZY). Two methods were used to determine the similarities between the two proteins: PSI Blast and Dali. The PSI Blast search function is a measure of the homology of primary structure between two proteins. It gives the measurement in terms of an E value; a value of less than 0.05 is significant. The E value for the D. melanogaster cryptochrome is 4x10-104, meaning the structures had extremely similar peptide sequences (6). The Dali Server uses a sum-of-pairs method to compare protein tertiary structures by analyzing intermolecular distances (7). Dali Server reports findings with a z-score; a z-score greater than 2 is considered significant. The D. melanogaster cryptochrome returned a z-score of 41.3 (7). The sequence of the D. melanogaster cryptochrome is similar in length with 534 residues, compared to 509 (4). Although the D. melanogaster cryptochrome contributes to the circadian rhythm in flies rather than DNA repair, it ultimately operates through the same method of using a flavin-adenine dinucleotide ligand to absorb light (8). The difference in applications is due to the fact that D. melanogaster has dedicated photolyases used for DNA repair while C. reinhardtii lacks photolyases so its cryptochrome is used instead (8). Therefore since the two proteins have similar primary and tertiary structures, their shared mechanism of FAD photoactivation is a result of their common structures. The only major structural difference between the two proteins is that the animal-like cryptochrome is a bilobal monomer while the D. melanogaster cryptochrome is a dimer composed of two nearly identical bilobal subunits (8). When compared to all other proteins in the photolyases/cryptochromes superfamily (PCSf), the animal-like cryptochrome from C. reinhardtii retains the distinctive tryptophan trio retained by all proteins in the family (1). In this specific protein, residues Trp-399, Trp-376, and Trp-22 compose the trio responsible for the characteristic electron transfer found in all members of the PCSf (1). The D. melanogaster cryptochrome has a similar group of tryptophans responsible for electron transfer in the residues Trp-342, Trp-397, and Trp-420 (8). 

The only product produced by the animal-like cryptochrome is the cleavage of the C6-C4 bond between two bases in DNA lesions. The FAD ligand directly influences this action (1). Phosphate stabilizes the target DNA while the other two ligands glycerol and 2-(N-morpholino)-ethanesulfonic acid occupy another antenna site so that FAD is the only antenna in use (1). The catalytic activity is a direct result of the protein’s previously mentioned critical residues as well as bilobal architecture that offers access to the centralized active site. Comparisons to a similar protein as well as the entire PCSf group indicate that the function of the active site is due to a group of three tryptophans that give all photolyases and cryptochromes their ability to utilize light for electron transfers (1). The strong connection between similarities in both primary and tertiary structure and function of cryptochromes indicate that their functionality is a direct result of their distinct structure. Further investigation into the mechanism of the cryptochrome could be useful for applications in cancer treatment in cases where UV light has caused DNA lesions. More research into the effect of the antenna ligand could prove useful for regulation of the circadian rhythm in other organisms based on the type of light used. The C. reinhardtii cryptochrome utilizes blue light, but replacing FAD with another ligand could change the type of light necessary for photoactivation. This could be used to understand how to accurately regulate the circadian rhythm of plants and animals to optimize activities such as sleep, hormone production, and cellular functions.