Jingjing Xu

Magnetoreception, the ability of animals to sense the Earth’s magnetic field, is a fascinating biological phenomenon. Cryptochromes, in particular cryptochrome 4a (CRY4a), have emerged as potential key players in mediating magnetic sensing in various bird species. Building on an earlier investigation of magnetic field effects on European robin ( Erithacus rubecula ) CRY4a, we focus here on CRY4a from the common/Japanese quail ( Coturnix coturnix/japonica ). Japanese quail is one of the very small number of domesticated bird species whose wild forms are migratory. A detailed spectroscopic study of purified quail CRY4a shows that it has magnetic properties similar to robin CRY4a, suggesting that the quail could be a promising additional experimental model with which to unravel the intricacies of magnetoreception in migratory birds.

Cryptochromes and photolyases are blue-light-sensitive flavoproteins that generally bind flavin adenine dinucleotide (FAD) and have distinct functions. Cryptochrome 4a (CRY4a) is a protein expressed in the double-cone photoreceptors of the retina in migratory songbirds like European robin ( Erithacus rubecula ) and is hypothesized as the primary sensor for avian magnetoreception. In addition to FAD, most photolyases and some cryptochromes bind antenna chromophores such as 8-hydroxy-5-deazaflavin (8-HDF) or 5,10-methenyltetrahydrofolate (MTHF) to enhance light absorption. Here, we investigated whether Erithacus rubecula Cryptochrome 4a ( Er CRY4a) also binds 8-HDF and/or MTHF. 8-HDF binding was studied by co-expressing Er CRY4a with the fbIC gene that encodes for 8-HDF synthase and thus for production of 8-HDF in E. coli . As a positive control for 8-HDF binding, we expressed Xenopus laevis 6-4 photolyase ( Xl 6-4PL) which is known to bind both FAD and 8-HDF. This experiment resulted in successful binding of 8-HDF to Xl 6-4PL, but not to Er CRY4a. We studied the binding of MTHF using in vitro reconstitution followed by UV-Vis spectroscopy and isothermal titration calorimetry (ITC) assays. No interaction was observed between MTHF and Er CRY4a. To theoretically understand the binding of potential antenna chromophores to Er CRY4a, we performed computational analyses. We found no similarity at the relevant binding sites between the sequences of Er CRY4a with proteins shown to bind MTHF or 8-HDF. This suggests that the binding pocket is not conserved. Our study proposes that Er CRY4a only harbor one light-sensitive cofactor, which in turn suggests a functional specialization different from most photolyases.

Cryptochrome 4a (Cry4a) is a magnetically sensitive protein that could enable night-migratory birds to sense the geomagnetic field for navigation. The key to the protein magnetic sensitivity is the flavin adenine dinucleotide (FAD) cofactor, which initiates the electron transfer within the protein leading to a spin-correlated radical pair. De-spite its importance, the mechanism of FAD binding in avian Cry4a proteins remains unclear. Here we show that point mutagenesis of positively charged arginine residue at position 356 to negatively charged glutamic acid completely depletes FAD binding from European robin ( Erithacus rubecula ) Cry4a. The result indicates that electrostatic in-teractions constitute the primary driving force that enables FAD binding in European robin Cry4a. The finding provides new structural insight into the molecular basis of FAD binding in Cry4 and advances our understanding on the biophysical underpinnings of bird magnetoreception. Table of Contents Graphic:

Avian cryptochrome 4 (Cry4) protein is a putative magnetosensitive molecule facilitating precise long-distance navigation in migratory birds. Two splice variants of Cry4 have been reported in the European robin ( Erithacus rubecula ), namely Er Cry4a and Er Cry4b. Er Cry4a protein is known to generate magnetically sensitive radical pairs for magnetoreception through electron transfer between the flavin adenine dinucleotide (FAD) cofactor and tryptophan residues within the protein. However, little is known about the Er Cry4b isoform. We therefore characterize the properties of Er Cry4b to determine whether it fulfils the prerequisites to be a radical-pair magnetic sensor molecule. Our results show that Er Cry4b protein does not bind FAD in vitro . Computational simulations reveal that the lack of FAD binding in Er Cry4b is probably due to protein structural dynamics. Furthermore, immunoprecipitation assays coupled with mass spectrometry suggest that Er Cry4b protein is undetectable in the robin retina, cerebellum, and liver. Meanwhile, transcript analyses show that ErCRY4b mRNA abundance is 10 times lower than that of ErCRY4a in the retina. In conclusion, Er Cry4b does not fulfil the prerequisites to be a radical pair-based magnetic sensing molecule due to the lack of FAD binding, and it might not even be expressed as a functional protein in the European robin.

Avian cryptochrome 4 (Cry4) protein is a putative magnetosensitive molecule facilitating precise long-distance navigation in migratory birds. Two splice variants of Cry4 were reported in European robin ( Erithacus rubecula ), namely Er Cry4a and Er Cry4b. It is known that Er Cry4a protein exhibits electron transfer between the flavin adenine dinucleotide (FAD) cofactor and tryptophan residues that generates magnetically sensitive radical pairs for magnetoreception. However, little is known about the Er Cry4b isoform. We therefore characterized the properties of Er Cry4b to see whether it fulfills prerequisites to be a radical pair magnetic sensor molecule. Our results show that Er Cry4b protein does not bind FAD in vitro . Computational structure simulations revealed that the FAD non-binding in Er Cry4b is likely due to protein structure dynamics. Furthermore, Er Cry4b protein abundance in the robin retina, cerebellum and liver is below the detection limit of immunoprecipitation assays coupled with mass spectrometry. Meanwhile, transcript analyses show that ErCRY4b mRNA abundance is 10 times less than ErCRY4b in the retina. In conclusion, Er Cry4b does not fulfill the prerequisites to be a radical pair based magnetic sensing molecule due to the lack of FAD binding, and it might not even be expressed as a functional protein in the European robin.

Cryptochrome 4a (Cry4a) has been proposed as the sensor at the heart of the magnetic compass in migratory songbirds. Blue-light excitation of this protein produces magnetically sensitive flavin–tryptophan radical pairs whose properties suggest that Cry4a could indeed be suitable as a magnetoreceptor. Here, we use cavity ring-down spectroscopy to measure magnetic field effects on the kinetics of these radical pairs in modified Cry4a proteins from the migratory European robin and from nonmigratory pigeon and chicken. B1/2, a parameter that characterizes the magnetic field-dependence of the reactions, was found to be larger than expected on the basis of hyperfine interactions and to increase with the delay between pump and probe laser pulses. Semiclassical spin dynamics simulations show that this behavior is consistent with a singlet–triplet dephasing (STD) relaxation mechanism. Analysis of the experimental data gives dephasing rate constants, rSTD, in the range 3–6 × 107 s−1. A simple “toy” model due to Maeda, Miura, and Arai [Mol. Phys. 104, 1779–1788 (2006)] is used to shed light on the origin of the time-dependence and the nature of the STD mechanism. Under the conditions of the experiments, STD results in an exponential approach to spin equilibrium at a rate considerably slower than rSTD. We attribute the loss of singlet–triplet coherence to electron hopping between the second and third tryptophans of the electron transfer chain and comment on whether this process could explain differences in the magnetic sensitivity of robin, chicken, and pigeon Cry4a’s.

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerization of avian Crys and the role it could play in the mechanism of magnetic sensing in migratory birds. Here, we present a combined experimental and computational investigation of the dimerization of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulfide bonds, chemical cross-linking, and photometric measurements show that disulfide-linked dimers are routinely formed, that their formation is promoted by exposure to blue light, and that the most likely cysteines are C317 and C412. Computational modeling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.