Cellular metabolites modulate in vivo signaling of Arabidopsis cryptochrome-1

Abstract

Cryptochromes are blue-light absorbing flavoproteins with multiple signaling roles. In plants, cryptochrome (cry1, cry2) biological activity has been linked to flavin photoreduction via an electron transport chain to the protein surface comprising 3 evolutionarily conserved tryptophan residues known as the 'Trp triad.' Mutation of any of the Trp triad residues abolishes photoreduction in isolated cryptochrome protein in vitro and therefore had been suggested as essential for electron transfer to the flavin. However, photoreduction of the flavin in Arabidopsis cry2 proteins occurs in vivo even with mutations in the Trp triad, indicating the existence of alternative electron transfer pathways to the flavin. These pathways are potentiated by metabolites in the intracellular environment including ATP, ADP, AMP, and NADH. In the present work we extend these observations to Arabidopsis cryptochrome 1 and demonstrate that Trp triad substitution mutants at W400F and W324F positions which are not photoreduced in vitro can be photoreduced in whole cell extracts, albeit with reduced efficiency. We further show that the flavin signaling state (FADH ) is stabilized in an in vivo context. These data illustrate that in vivo modulation by metabolites in the cellular environment may play an important role in cryptochrome signaling, and are discussed with respect to possible effects on the conformation of the C-terminal domain to generate the biologically active conformational state.

Electron spin relaxation can enhance the performance of a cryptochrome-based magnetic compass sensor

“…Despite the finding that [FAD •− + ] TrpH C • in isolated AtCry1 in vitro responds to relatively weak magnetic fields [23], it is not clear that this radical pair would necessarily be the sensor in an in vivo cryptochrome magnetoreceptor. Protein-protein and protein-ligand interactions in a cellular environment could result in a different, and possibly more sensitive, radical pair via an alternative or extended electron transfer pathway [44,54,55]. It is therefore important to ask whether Nature could have contrived a cryptochrome-based magnetic sensor in which the exchange and dipolar interactions are so weak that they do not adversely affect its performance.…”

ATP boosts lit state formation and activity of Arabidopsis cryptochrome 2

“…The similarity in sequence and structure between DNA photolyases and cryptochromes is restricted to the N-terminal domain of cryptochromes; this photolyase-homology region (PHR) consists of an N-terminal a/b domain and a C-terminal a-domain, which accommodates the FAD cofactor in a Ushaped conformation typical of all family members (Brautigam et al, 2004;Chaves et al, 2011). In contrast to photolyases, cryptochromes have a C-terminal extension (CCE) that varies in length and sequence among cryptochromes; the CCE is engaged in signal transduction together with the PHR domain (El-Esawi et al, 2015;Th€ oing et al, 2015).…”

Nicotinamide Adenine Dinucleotides Arrest Photoreduction of Class II DNA Photolyases in FADH˙ State

“…Furthermore, recombinant At Cry1 and At Cry2 form the active signaling state FADH ˙ upon illumination despite a disrupted tryptophan cascade, when analyzed in insect cells. Likewise, it was also reported for a variant of the tryptophan triad of At Cry2 that small metabolites like NADH, NADPH and even derivatives of ATP are able to enhance the accumulation of FADH ˙ .…”

“…For these variants no photoreduction was observed in vitro , but 10 ‐ 20 % of the activity remained in vivo . However, the formation of the active state FADH ˙ was observed for these variants in insect cells . From these findings an alternative electron transfer pathway was suggested independent of the tryptophan cascade under physiological conditions for At Cry1 and At Cry2 .…”