Light-Induced Conformational Changes of Cyanobacterial Phytochrome Cph1 Probed by Limited Proteolysis and Autophosphorylation † Berta Esteban, ‡ Montserrat Carrascal, § Joaquin Abian, § and Tilman Lamparter* ,‡ Pflanzenphysiologie, Freie UniVersita ¨t Berlin, Ko ¨nigin Luise Strasse 12-16, D-14195 Berlin, Germany, and Structural and Biological Mass Spectrometry Unit, IIBB-CSIC, IDIBAPS, Rosellon 161, 7 Planta E-08036 Barcelona, Spain ReceiVed July 22, 2004; ReVised Manuscript ReceiVed October 14, 2004 ABSTRACT: Photoreceptor chromoproteins undergo light-induced conformational changes that result in a modulation of protein interaction and enzymatic activity. Bacterial phytochromes such as Cph1 from the cyanobacterium Synechocystis PCC 6803 are light-regulated histidine kinases in which the light signal is transferred from the N-terminal chromophore module to the C-terminal kinase module. In this study, purified recombinant Cph1 was subjected to limited proteolysis using trypsin and endoproteinase Glu-C (V8). Cleavage sites of chromopeptide fragments were determined by MALDI-TOF and micro-HPLC on-line with tandem mass spectrometry in an ion trap mass spectrometer. Trypsin produced three major chromopeptides, termed F1 (S56 to R520), F2 (T64 to R472), and F3 (L81 to R472). F1 was produced only in the far-red absorbing form Pfr within 15 min and remained stable up to >1 h; F2 and F3 were obtained in the red-light absorbing form Pr within ca. 5-10 min. When F1 was photoconverted to Pr in the presence of trypsin, this fragment degraded to F2 and F3 within 1-2 min. On size exclusion chromatography, F1 eluted as a dimer in the Pfr and as a monomer in the Pr form, whereas F2 and F3 behaved always as monomers, irrespective of the light conditions. These and other results are discussed in the context of light-dependent subunit interactions, in which amino acids 473-520 within the PHY domain are required for chromophore-module subunit interaction within the homodimer. V8 proteolysis yielded five major chromopeptides, F4 (T17 to N449), F5 (T17 to E335), F6 (T17 to E323), F7 (unknown sequence), and F8 (tentatively L121 to E323). F6 and F8 were formed in the Pr form, whereas F4, F5, and F7 were preferentially formed in the Pfr form. Three amino acids next to specific cleavage sites, R520, R472, and E323, were altered by site-directed mutagenesis. The mutants were analyzed by UV- vis spectroscopy, size exclusion chromatography, and autophosphorylation. Histidine kinase activity was low in R472A, R520P, and R520A; in all mutants, the ratio of phosphorylation intensity between Pr and Pfr was reduced. Thus, light regulation of autophosphorylation is negatively affected in all mutants. In R472P, E323P, and E323D, the phosphorylation intensity of the Pfr form exceeded that of the wild-type control. This result shows that the histidine kinase activity of Cph1 is actively inhibited by photoconversion into Pfr. Phytochromes are widely distributed biliprotein photo- receptors that are most sensitive in the red or far-red region of the visible spectrum (1). The photocycle of typical phytochromes has two thermostable, spectrally different forms that are interconverted by light (2). Phytochromes are synthesized in the red-absorbing form, Pr, in a process during which the chromophore becomes autocatalytically attached to a conserved cysteine residue. Photoconversion into Pfr, the far-red absorbing form, is initiated by a rapid isomer- ization around the C15dC16 double bond of the chromo- phore (3-5). Thereafter, thermal relaxations into Pfr are observed in the microsecond and millisecond time range (6). Typical phytochrome proteins consist of an N-terminal chromophore module and a C-terminal regulatory module. Within the chromophore module, computer algorithms identify two to three different subdomains. In some bacterial phytochromes, such as Cph1 from the cyanobacterium Synechocystis PCC 6803, a PAS domain is found close to the N-terminus of the protein (see Figure 1C for the domain structure of Cph1). In biliverdin-binding phytochromes of proteobacteria, the homologous region bears the chromo- phore-binding cysteine residue (7). The GAF domain is the region of the highest homology among phytochromes. The chromophore-binding cysteine of cyanobacterial and plant phytochromes, which incorporate phycocyanobilin or phyto- chromobilin, lies within this domain. It has been shown for recombinant fragments of Cph2, a nontypical phytochrome from Synechocystis, that the GAF domain is sufficient for the chromophore ligation reaction (8). The C-terminal subdomain of the chromophore module is a so-called PHY domain, which is probably important for spectral integrity. Whereas the entire chromophore module is spectrally identical with the full-length protein (6, 9, 10), truncated proteins which lack part of the PHY domain are † This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 498, Teilprojekt B2. * Corresponding author. Tel: +49 (0)30 838 54918. Fax: +49 (0)- 30 838 84357. E-mail: lamparte@zedat.fu-berlin.de. ‡ Freie Universita ¨t Berlin. § IIBB-CSIC, IDIBAPS. 450 Biochemistry 2005, 44, 450-461 10.1021/bi0484365 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/16/2004