Hyperactivity of the Arabidopsis cryptochrome (cry1) L407F mutant is caused by a structural alteration close to the cry1 ATP-binding site

J Biol Chem. 2017 Aug 4;292(31):12906-12920. doi: 10.1074/jbc.M117.788869. Epub 2017 Jun 20.

Abstract

Plant cryptochromes (cry) act as UV-A/blue light receptors. The prototype, Arabidopsis thaliana cry1, regulates several light responses during the life cycle, including de-etiolation, and is also involved in regulating flowering time. The cry1 photocycle is initiated by light absorption by its FAD chromophore, which is most likely fully oxidized (FADox) in the dark state and photoreduced to the neutral flavin semiquinone (FADH°) in its lit state. Cryptochromes lack the DNA-repair activity of the closely related DNA photolyases, but they retain the ability to bind nucleotides such as ATP. The previously characterized L407F mutant allele of Arabidopsis cry1 is biologically hyperactive and seems to mimic the ATP-bound state of cry1, but the reason for this phenotypic change is unclear. Here, we show that cry1L407F can still bind ATP, has less pronounced photoreduction and formation of FADH° than wild-type cry1, and has a dark reversion rate 1.7 times lower than that of the wild type. The hyperactivity of cry1L407F is not related to a higher FADH° occupancy of the photoreceptor but is caused by a structural alteration close to the ATP-binding site. Moreover, we show that ATP binds to cry1 in both the dark and the lit states. This binding was not affected by cry1's C-terminal extension, which is important for signal transduction. Finally, we show that a recently discovered chemical inhibitor of cry1, 3-bromo-7-nitroindazole, competes for ATP binding and thereby diminishes FADH° formation, which demonstrates that both processes are important for cry1 function.

Keywords: ATP; cryptochrome; nucleotide; nucleotide binding; photobiology; plant biochemistry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism*
  • Amino Acid Substitution
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / antagonists & inhibitors
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Binding Sites
  • Binding, Competitive
  • Biocatalysis
  • Cryptochromes / antagonists & inhibitors
  • Cryptochromes / chemistry
  • Cryptochromes / genetics
  • Cryptochromes / metabolism*
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Hot Temperature / adverse effects
  • Indazoles / chemistry
  • Indazoles / metabolism
  • Indazoles / pharmacology
  • Ligands
  • Models, Molecular*
  • Molecular Docking Simulation
  • Mutagenesis, Site-Directed
  • Mutation*
  • Peptide Fragments / antagonists & inhibitors
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Conformation
  • Protein Denaturation
  • Protein Interaction Domains and Motifs
  • Protein Stability
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Structural Homology, Protein

Substances

  • 3-bromo-7-nitroindazole
  • Arabidopsis Proteins
  • CRY1 protein, Arabidopsis
  • Cryptochromes
  • Enzyme Inhibitors
  • Indazoles
  • Ligands
  • Peptide Fragments
  • Recombinant Proteins
  • Adenosine Triphosphate

Associated data

  • PDB/1U3D