Absence of 8-HDF and MTHF Antenna Chromophore Binding in Er CRY4a Suggests a Possible Flavin-Only Cofactor State: Insights from Biochemical and Computational Analyses

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.