Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes

doi:10.1073/pnas.0809180106

Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes

^aGraduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan;
^bDepartment of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
^cLife Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;
^dRegulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; and
^eRadiation Biology Center, Kyoto University, Kyoto 606-8501, Japan

Edited by Joanne Chory, The Salk Institute for Biological Studies, La Jolla, CA, and approved February 9, 2009 (received for review October 2, 2008)

Abstract

Homologous flavoproteins from the photolyase (PHR)/cryptochrome (CRY) family use the FAD cofactor in PHRs to catalyze DNA repair and in CRYs to tune the circadian clock and control development. To help address how PHR/CRY members achieve these diverse functions, we determined the crystallographic structure of Arabidopsis thaliana (6-4) PHR (UVR3), which is strikingly (>65%) similar in sequence to human circadian clock CRYs. The structure reveals a substrate-binding cavity specific for the UV-induced DNA lesion, (6-4) photoproduct, and cofactor binding sites different from those of bacterial PHRs and consistent with distinct mechanisms for activities and regulation. Mutational analyses were combined with this prototypic structure for the (6-4) PHR/clock CRY cluster to identify structural and functional motifs: phosphate-binding and Pro-Lys-Leu protrusion motifs constricting access to the substrate-binding cavity above FAD, sulfur loop near the external end of the Trp electron-transfer pathway, and previously undefined C-terminal helix. Our results provide a detailed, unified framework for investigations of (6-4) PHRs and the mammalian CRYs. Conservation of key residues and motifs controlling FAD access and activities suggests that regulation of FAD redox properties and radical stability is essential not only for (6-4) photoproduct DNA repair, but also for circadian clock-regulating CRY functions. The structural and functional results reported here elucidate archetypal relationships within this flavoprotein family and suggest how PHRs and CRYs use local residue and cofactor tuning, rather than larger structural modifications, to achieve their diverse functions encompassing DNA repair, plant growth and development, and circadian clock regulation.

Footnotes

²To whom correspondence should be addressed. E-mail: edg{at}scripps.edu
Author contributions: K.H., T.T., J.A.T., S.P., and E.D.G. designed research; K.H., L.D., A.S.A., J.Y., and S.-T.K. performed research; S.I. contributed new reagents/analytic tools; K.H., L.D., A.S.A., J.A.T., S.I., S.P., and E.D.G. analyzed data; and K.H., S.P., and E.D.G. wrote the paper.
↵¹Present address: Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates have been deposited in in the Protein Data Bank, www.pdb.org (PDB ID code 3FY4).
This article contains supporting information online at www.pnas.org/cgi/content/full/0809180106/DCSupplemental.