title: Light-induced conformational changes of cyanobacterial phytochrome Cph1 probed by limited proteolysis and autophosphorylation.
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.
authors.affiliation: Pflanzenphysiologie, Freie Universität Berlin, Königin Luise Strasse 12-16, D-14195 Berlin, Germany.
authors.forename: Berta
authors.initials: B
authors.lastname: Esteban
authors.forename: Montserrat
authors.initials: M
authors.lastname: Carrascal
authors.forename: Joaquin
authors.initials: J
authors.lastname: Abian
authors.forename: Tilman
authors.initials: T
authors.lastname: Lamparter
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000071677:Histidine Kinase; D006868:Hydrolysis; D008027:Light; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D020283:Photoreceptors, Microbial; D010834:Phytochrome / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D046939:Synechocystis; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins; D010834:Phytochrome; D011494:Protein Kinases; D000071677:Histidine Kinase; C413623:Cph1 phytochrome protein, bacteria; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin