Animal Type 1 Cryptochromes

Abstract

It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD . , in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FAD ox when purified, and the flavin is readily reduced to FAD . by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD . in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD . represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs.Cryptochromes are photolyase-related flavoproteins that play important roles in regulating the circadian clock in animals and growth and development in plants (1-3). The mechanism of photosignaling by animal cryptochromes is not known. Previously, it was thought that CRYs 3 in Drosophila and other insects function as circadian photoreceptors and in mouse and other vertebrates function as core components of the molecular clock (4). Recently, this view was revised when it was realized that some insects such as the honeybee possess only a mammalian CRY-like cryptochrome and others such as the monarch butterfly possess both Drosophila CRY-like and mammalian CRY-like cryptochromes (5, 6). It was proposed that Drosophila-like CRYs should be referred to as Type 1 CRYs and the mammalian-like CRYs should be referred to as Type 2 CRYs (6). Furthermore, it was found that all Type 1 CRYs tested were subject to light-induced proteolysis in Schneider 2 (S2) cells and, hence, were considered to function as circadian photoreceptors in a manner analogous to DmCRY (6). Similarly, it was shown that insect Type 2 CRYs, like the mammalian CRYs, functioned as core clock proteins with no demonstrable photoreceptor activity (6).We are interested in the photoreceptor function of CRY and specifically in the cryptochrome photocycle. Type 1 CRYs are well suited for this purpose because their photoinitiated proteolysis constitutes a convenient functional assay (7-9). Two recent studies reported that Type 1 CRYs from Drosophila melanogaster and the monarch butterfly (Danaus plexippus), purified as recombinant proteins, contained near-stoichiometric amounts of flavin in the two-electron oxidized, FAD ox , form. Exposure of these CRYs to light reduced the flavin to the flavin anion ...

Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase

“…However, we do have a quantum yield for an enzymatic reaction that is initiated by photoexcited Type 1 CRYs, the proteolytic degradation of the CRY itself. 17 The quantum yield for this process is in the range of φ~10 -3 , which is clearly within the range of theoretical maximum of about φ~0.2, assuming conventional photochemical reactions. In fact, proteolysis is at least one and perhaps more than one step removed from the primary photochemical event and hence we expect that the actual quantum yield of electron transfer (if that is the mechanism) of Type 1 CRYs may approach the theoretical limit.…”

“…17 The redox status of FAD photoreduction, the Trp triad and the effects of Cys mutations at the flavin binding site on the flavin photochemistry are listed in Table 1. To put these results in perspective we summarize our recent functional studies in which light-induced proteolysis of Type 1 CRYs in vivo was used as an endpoint for photoreceptor activity: 16,17 The C→N mutants showed the same photoinduced proteolysis as the wild type CRYs, excluding the possibility of photoadduction with the cysteine near the N5 position as the initial signaling steps, which has been observed in phototropin, 24,25 and suggests that FADHfunctions as well as FAD •-in insect Type 1 CRYs. In contrast, the C→A mutants in which flavin is readily photoreduced to FAD •-but reoxidizes within seconds (more than an order of magnitude faster than their wild-type counterparts) showed no photoreceptor activity in vivo , 17 indicating that a stable FAD •-but not oxidized FAD must be the active form of flavin in …”

“…16,17 Final protein concentrations of 20-50 μM in 50% (v/v) glycerol in a buffer of 50 mM sodium phosphate, 100 mM NaCl, pH 7.4 or of 50 mM Tris-HCl, 100 mM NaCl, 1mM EDTA, 5 mM dithiothreitol, pH 7.5 were used in femtosecond-resolved studies. The purifications of EcPhr and At(6-4) have been described in detail previously with some modifications.…”

Light-activated Cryptochrome Reacts with Molecular Oxygen to Form a Flavin–Superoxide Radical Pair Consistent with Magnetoreception

“…Similarly for Drosophila cryptochrome, the resting state of cryptochrome has been shown to be the oxidized form; this photoreaction occurs in living insect cells and has been documented by independent techniques (13). A suggestion that illumination of radical in DmCRY may lead to activation by an unknown mechanism (29,30) is not in contradiction to these results as formation of the radical would still require photoreduction of oxidized flavin and be opposed by flavin re-oxidation. Therefore, an important effect of oxygen is implied in the photoreactions of both animal and plant cryptochromes, and conclusions from this study can be generalized to all cryptochromes that undergo a reoxidation of flavin to FAD ox in darkness subsequent to illumination.…”

Structure and Function of Photolyase and in Vivo Enzymology: 50th Anniversary

“…The mechanisms of photoreception and magnetoreception by CRYs are not understood at present. Even the redox state of FAD in plant and insect CRYs is a matter of considerable debate (45)(46)(47), although the majority of action spectrum studies favor photolyase-like FADH Ϫ or FAD . states (44,48).…”