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

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: phosphatebinding 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.blue-light photoreceptor ͉ circadian clock ͉ electron transfer ͉ flavoprotein ͉ FAD

Crystal structure of a prokaryotic (6-4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore

“…His366 in PhrB is stabilized by van der Waals contacts with Leu370 and Met410 (Fig. 3A), whereas the homologous His in eukaryotic photolyases is stabilized by a hydrogen bonding network (15,22). The orientation of His366 in PhrB, however, matches with the homologous His residue of Drome (6-4) PL (Fig.…”

“…In a structural alignment (23) of PhrB and two eukaryotic (6-4) photolyases (15,22), we have identified 34 identical amino acids, 10 of which are conserved to 99-100% in all FeS-BCPs. Three of these amino acids cluster together in the His365-His366-X-X-Arg369 motif.…”

Identification of a Novel Cryptochrome Differentiating Domain Required for Feedback Repression in Circadian Clock Function

“…Our findings shed new light on the functional importance of the CRY1-PHR(313-426) and the CTD in the clock mechanism. Several previous studies identified a subset of common motifs and sites, including nuclear localization sequences, coiled-coils, phosphorylation sites of CK1⑀, GSK3␤, MAP kinase, and AMP-activated protein kinase (44,45,47,49,50,(53)(54)(55)(56), and surely additional motifs remain yet to be identified. The functional significance of these various sequences and structural features in CRY function will need to be tested using the assays developed in this study.…”

“…Based on known structure and domain functions of PHL/CRY proteins (18,19,44), we divided CRY1 and -2 proteins into four regions, namely A, B, C, and D ( Fig. 2A; supplemental Fig.…”

Recognition and Repair Pathways of Damaged DNA in Higher Plants

“…The structure and reaction mechanisms of photolyases have been intensively studied in the last decade, providing us with plentiful data on their function. Photolyases have two types of chromophoric co-factors that are used for photoreactivation (Huang et al, 2006;Ozturk et al, 2008;Hitomi et al, 2009). One chromophore is FADH -, the two electron reduced form of FAD, while the second one can be either methenyltetrahydrofolate (MTHF) or 7,.…”