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Plant UVR8 Photoreceptor Senses UV-B by Tryptophan-

Cross-Dimer Salt Bridges

John M.Christie,1,2Andrew S.Arvai,2Katherine J.Baxter,1*MonikaHOHara,1Sharon M.Kelly,1MichaelHothorn,3Brian O.Smith,1KenicElizabeth D.Getzoff21Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary an

University of Glasgow, Glasgow G12 8QQ, UK. 2Department of Molecular Biology The Scripps Research Institute, La Jolla, California, USA. 3Plant Biology LaboratoryLa Jolla, California, USA. 4Life Science Division, Lawrence Berkeley National Laboof Laboratory Equipment, National Institute of Biomedical Innovation, 7-6-8, Saito-A

*Th th t ib t d ll t thi k

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blade is contiguous in sequence with the N- and C-termini inthe first and last blades, respectively (figs. S1 and S4),potentially permitting greater conformational flexibility.

Each doughnut-shaped UVR8 monomer is 40-50 in

diameter, by ~35 high with a central water-filled tunnel(Fig. 1B). Crystal packing of the trypsin-treated protein,which remains UV-B-responsive (fig. S5), suggests twopotential cylindrical dimer assemblies (Fig. 1C, inset). Smallangle x-ray scattering (SAXS) (table S2) clearly distinguishesthe correct dimer in solution (Fig. 1C), by agreement betweencalculated and observed scattering profiles (1113). Thesmaller, concave surfaces of the two subunits assemble face-

to-face, but offset by about 10 (Fig. 1, B and D). The two-fold dimer symmetry juxtaposes different blades from eachsubunit, except self-pairing blade 4 (Fig. 1B). SAXS results(Fig. 1D) show different lengths, but the same overall shapefor full-length UVR8 and the trypsin-cleaved protein used for

ll h l l l d i d f S S

D96 (UVR8D96N/D

bridges (UVR8R28

monomers (Fig. 2Ethe R286 salt bridg

In each monomresidues dominateconserved Gly-Trp6, and 7 (fig. S1) gtryptophans, W23UVR8 photorecepprotruding tight tuoutwards, and His

buried ring (Fig. 1adjacent Arg guanorientation and pa3A). R234 and R3W285 and W233. bl d 4 5 d 6 (

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photoperception, with W285 as the principal UV-B sensor.W233 is also important, not only in photoreception butparticularly in maintaining exciton coupling, whereas W337and W94 play auxiliary roles.

To examine the in vivo role of W285 in UV-B perception,we expressed GFP-UVR8W285A in mutant uvr8-1 plants andassayed UV-B induction ofHY5 transcripts, mediated byUVR8 (1). GFP-UVR8W285A does not restore UV-B-mediatedinduction ofHY5 to uvr8-1 mutants (Fig. 4A). However, lossof activity in the W285A mutant does not result from grossstructural changes; SAXS analysis of UVR8W285A confirmedthat the dimer protein has dimensions similar to those of the

wild type (Fig. 4B). Thus, these experiments show the in vivofunctional importance of W285 in UV-B photoreception.Remarkably, although UVR8W285F is unable to respond toUV-B, it does respond to UV-C, consistent with the shorterwavelength absorption of phenylalanine compared to

predominantly W2transfer of an exciTrp pyramid to adjneutralization, con

bridges and thus d4F). The complex aromatic cluster suinterconnectivity otogether the dimerrobust, concerted msignaling.

In conclusion, w

known photorecepits intrinsic tryptopThe presence of pumosses suggests thpromote plant survll d hi h l

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10. Materials and methods are available as supporting

material on Science Online.11. G. L. Hura et al., Robust, high-throughput solution

structural analyses by small angle x-ray scattering

(SAXS).Nat Methods6, 606 (2009).12. C. D. Putnam, M. Hammel, G. L. Hura, J. A. Tainer, X-

ray solution scattering (SAXS) combined withcrystallography and computation: defining accuratemacromolecular structures, conformations and assembliesin solution. Q Rev Biophys40, 191 (2007).

13. N. Nishimura et al., Structural Mechanism of AbscisicAcid Binding and Signaling by Dimeric PYR1. Science

326, 1373 (2009).14. E. Krissinel, K. Henrick, Inference of macromolecularassemblies from crystalline state.J Mol Biol372, 774(2007).

15. I. B. Grishina, R. W. Woody, Contributions of tryptophanid h i h i l di h i f l b l i

collected at SIBAnalysis TechnDepartment of Environmental

structure factorwith accession

Supporting Onlin

www.sciencemagMaterials and MetFigs. S1 to S8Tables S1 to S3

References (203819 December 201Published online 9

Fig 1 Structure of

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UVR8W233F and UVR8W233A; (E) UVR8W285F and UVR8W285A;(F) UVR8W337F and UVR8W337A; (G) triple mutantsUVR8W233F/W285F/W337F and UVR8W233A/W285A/W337A.

Fig 4. Key features of UVR8 photoreceptor mechanism(A)qRT-PCR analysis of UV-B induction ofHY5 transcripts inwild-type, uvr8-1, and uvr8-1 expressing GFP-UVR8W285A(line 7-1). Inset shows Western blot of GFP-UVR8 in plantlines and Ponceau S staining of rbcL protein as loadingcontrol. (B) SAXS Pair-distance-distribution functions [P(r)]show that UVR8W285A is dimeric, with only subtleconformational differences from wild-type. (C)Far-UV CDspectra of UVR8W285F before and after exposure to 40 min

UV-C. (D) Size exclusion chromatography of UVR8W285Fmutant protein exposed to UV-C (solid and dotted magentalines).(E) Far-UV CD spectra of UVR8R146A/R286A,UVR8R286A/R338A and UVR8D96N/D107N mutants. (F)Model forUV-B photoreception by UVR8. UV-B sensing (by Trp

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