The 3D structure solution of the photosensory module from the cyanobacterial phytochrome Cph1 forms the basis to elucidate the photoconversion and the signalling mechanism of canonical phytochromes. The first crystal structure of the sensor module, comprising the PAS, GAF and PHY domains, from Cph1 in its Pr state was solved at 2.45 Å resolution. 2VEA confirmed the figure-of-eight knot, produced by an N-terminal extension from the PAS domain passing through a protruding loop of the GAF domain. Furthermore, the structure shows that the GAF and PHY domains are related thus placing phytochromes among tandem GAF proteins. Unlike previous structures of incomplete bidomains from bacteriophytochromes, 2VEA displays a completely closed chromophore pocket, sealing of the chromophore cavity being accomplished by a tongue-like protrusion from the PHY domain. Hence, 2VEA provides structural evidence for the necessity of the PHY domain to have a photochemically competent phytochrome. Several key residues were identified and selected for site-directed mutagenesis based on 2VEA that might be involved in either the photoconversion or the signalling mechanism. 48 different mutants were expressed, purified and characterised by UV-Vis spectroscopy. Selected mutants underwent further investigation (fluorescence spectroscopy, SEC, kinetic studies and crystallisation) to determine their function in phytochromes. Three tyrosines close to the D-ring (Tyr-176, Tyr-203 and Tyr-263) of the chromophore were identified to play a significant role in the photoconversion mechanism. This work confirmed the importance of Tyr-176 which was analysed in detail in previous studies. Furthermore Tyr-203 mutations showed that this residue is also essential for effective photoconversion. Studies on Tyr-263 mutants suggest that its aromatic character is essential for photoconversion, but that the hydroxyl group only improves the quantum yield of phototransformation. The 3D structure of Y263F solved at 1.95 Å resolution gave new insights into the heterogeneity of phytochrome structures; apart from the PHY domain shift in relation to the PAS-GAF lobe enabled by a straightening of the connecting &
#945;9 helix, the attachment site of the chromophore as well as several residues in the pocket displayed two possible conformations. Based on the results obtained by UV-Vis as well as fluorescence spectroscopy, the primary step in the photoconversion mechanism is most likely the isomerisation of the D-ring.This work also identified key residues interacting with the propionate side chains of the chromophore in either the Pr or Pfr states which might transfer the signal from the chromophore to the transmitter module. Furthermore, conserved residues belonging to the PRxSF motif within the tongue of the PHY domain may be involved in the signalling mechanism. This work suggests that both the tongue and the propionate side chains act in conjunction to transmit the signal from the sensor to the transmitter module by shifting the PHY domain.The crystal structure of the sensor module in its Pr state and the characterisation of conserved residues within the GAF and PHY domains constitute a crucial step in the elucidation of the photoconversion and signalling mechanism. Solving the structure of either the Pfr state of a canonical phytochrome or the full length photoreceptor comprising both the sensor and the transmitter modules might not only confirm but also improve the proposed models of photoconversion and phytochrome signalling in this work.
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