The biological function of Cryptochrome 3 (Cry 3) found in Arabidopsis Thaliana is not yet fully understood (4). However, it is known that Cry 3 is important in maintaining circadian rhythm, and that it is transported into chloroplast and mitochondria suggesting that Cry3 serves some function in these organelles (8). Cry 3 is similar to other cryptochromes in that it uses blue light to carry out its enzymatic functions and possess two key ligands: flavin adenine dinucleotide (FAD) and 5,10-methylenetetrahydrofolate (MTHF) (4). An insight on the possible function of Cry 3 can come from other cryptochromes and homologous proteins. In other species, such as Synechocystis, DASH Cryptochromes seem to serve as transcriptional repressors (5). Other evidence also shows Cry 3 possess an affinity towards DNA which further suggests that it may interact with nucleic acids to some degree (8).
Even though its
exact function and role is not yet fully known, the protein’s structure and the
purpose of various residues and ligands have been determined. Cry 3 is a
globular protein which has 525
residues and a molecular weight of 60.2 kDA with a theoretical pI of 8.98 (3). Furthermore,
the 45% of the protein is helical and 8% of the protein consists of beta sheets
(7). Although Cry 3 is mostly in its monomer state in solution, and the dimer
is not common and thus not significant in vivo, the crystal structure has been
determined in its dimer state(8). A unique characteristic of Cry 3 is
that it possesses an N-terminal extension of the protein that is usually found
on the C-terminal side of Cry 1 and Cry 2 (8). This N1-G40 N-terminal extension
of Cry 3 possesses three common features found in the C-terminal side of Cry 1
and Cry 2:a conserved aspartic residue region, a string of acidic residues, and a string of serine residues (8).
In cry 1 and cry 2, this N-terminal extension has been known to be part
in light-dependent signaling, but this function has not been confirmed in Cry 3
(9). The C-terminal in Cry 3 is comprised of “10 glycines and other polar
residues”, so it is likely that this end portion of protein has no structure (8).
The Cry 3 protein possesses a FAD cofactor and MTHF antenna chromophore, which aid in driving electron
transfer by gathering energy from light (6). These electron transfers are vital
for carrying out the biological function of the Cry 3 protein similar to other
cryptochromes and the closely related DNA photolyase proteins. The redox state of FAD and light intensity
influences the photocycles of MTHF, which is known to aid in transferring
energy to FAD (8). The MHTF and FAD cofactors are non-covalently bonded to the protein,
but are bound by hydrogen bond interactions on key residues (6,7).
The binding pocket of MTHF is made from three alpha helices and three loops. The MTHF chromophore is located between the N-terminal antenna and the C-terminal FAD binding domain and “seals thereby an internal water-filled cavity along the domain interface” (8). The close proximity of MTHF and FAD created by this conformation dramatically increase the rate MTHF transfers energy to FAD. Therefore, the overall efficiency of energy transfer is increased, which then increases the efficiency of Cry3 (9). The residues that hydrogen bond with MTHF and create the binding pocket is highly conserved among the DASH Cry (4). By analyzing the MTHF binding pocket of similar proteins, the importance of certain residues of MTHF binding can be highlighted, such as the closely related photolyase family (4). When the MTHF binding pocket of E. coli photolyase was compared to Cry 3, E. coli photolyases only had 8 residues that interacted, while Cry 3 had 11 (2). These two proteins shared only shared one homologous residue: E149 (8). E149 hydrogen binds with the nitrogen-3 (N3) in the pterin group and the carbon-2 (C2) in the amino group of MTHF (4). The importance of this residue is further highlighted by studies that show Cry 3 that lack this E149 show a significant change in absorption and fluorescence (4). The pattern created is more common with a protein bound to a fully oxidized FAD suggesting a significant change in the MTHF’s ability to transfer energy to FAD (4).
FAD is surrounded
by a pocket of basic residues and contains 12 conserved residues. The binding pocket of FADconsists of 6 alpha helices and 3 loops. A unique
residue found in cry 3, but not in cry 1 and cry 2 is N428. N428 hydrogen bonds with itself on its carobxamide side chain to the nitrogen-5 (N5) of FAD (8).The electron transfer chain in cry 3 is also thus highly conserved and starts
from the protein surface to FAD. The chain follows thus: W356, W409, W432, and FAD (10). Y423 and Y429 are also thought to be part of this
process since these residues contact the MTHF chromophore. Y423 is in hydrogen
bonding distance to the nitrogen atoms of MTHF. These residues may also play an
important role in regulating the spectral changes of MTHF via causing changes
in the MTHF binding sites by light driven changes of the redox state of FAD
(4). The FAD itself exists in three different states: a fully oxidized state, a
semiquinone state, and two reduced states where protons are added to the nitrogen
atoms (10). The different states of FAD can be seen in images (10). Thus, MTHF
helps to absorb ultraviolet radiation in order to transfer to the FAD chromophore
and therefore turning FAD into its fully reduced state.
Cry 3, a member of
the Crypto_Dash family, is very similar to proteins in the DNA photolyase
superfamily. DNA photolyases also possess FAD and MTHF (4). Furthermore, the
FAD bound to the Cry 3 is in a “U-shaped” conformation, which is also evident in
the DNA photolyase family (6). Although both DNA photolyases and DASH
cryptochromes utilize blue light and FAD cofactors, DASH cryptochromes regulate
the circadian rhythm of an organism and various growth and adaptive functions,
while DNA photolyases are vital in DNA repair (4). This remarkable level of
homology explains why cryptochromes also possess an affinity to DNA. Since Cry
3 can bind to DNA, another explanation is required to explain the functional
differences between Cry 3 and photolyases (6).
Substrate competition can be an explanation for the functional
differences between Cry 3 and class 1 cyclobutane pyrmidine dimer (CPD) photolyases,
which repairs DNA by returning CPD’s into their original state (6). The
hydrophobic cavity found in CPD photolyases, essential in substrate bonding, is
charged in Cry3, decreasing the spontaneous binding to the DNA substrate (6). The charged surface are contributed by E444, R443, R446, and Q395consist of or
are exposed to the CPD binding site which is responsible for the lower affinity
(6).
An E value of less
than 0.05 is considered to be significantly homologous to a protein, and an E
value of 0 indicates a protein with the same primary structure. A BLAST search
of Cry 3 found the E value of DNA photolyases of Synechosystis to be 2e-160 (1). This extremely miniscule value is a
mathematical representation on how similar photolyases and cryptochromes are. Furthermore,
a Dali Search also found a Z value of 48.4 and an rmsd value of 1.6 (5). By
analyzing similar proteins, the nature and functions of the protein of
interests can be elucidated. The exact purpose of these chromophores and
electron chain transfer is unknown in the function of Cry 3, which is also
still being determined. However, the high conservation of key residues and the
function of homologous proteins suggest that these chromophores and the
electron chain transfer are vital. In photolyases, the fully reduced FAD
catalyzes a transfer of an electron radical to the unstable pyrimidine dimer
resulting in a spontaneous decomposition into two pyrmidines (8). The superposition of one of Cry 3 dimers and Subunit B of DNA photolyases of Synechosystisalso demonstrates the overall structural
similarities of these two proteins.