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Phytochromes are photoreceptors that incorporate a bilin chromophore and undergo a photoconversion between a red light (Pr) and a far red light (Pfr) absorbing state. SynCph2 from Synechocystis sp. PCC 6803 comprises the domain architecture GAF1-GAF2-GGDEF1*-EAL-GAF3-GGDEF2, which indicates that it is a hybrid of a GAF-GAF bidomain phytochrome and a cyanobacteriochrome, represented by the GAF3 domain.
This work provides a detailed analysis of the N-terminal GAF1-GAF2 module. SynCph2(1-2) retains the red/far red photochemistry of canonical phytochromes, therefore, the GAF2 domain is able to substitute for the PHY domain in the latter. The PCB chromophore is covalently attached to Cys-129 in the GAF1 domain. In cooperation with the group of P. Hildebrandt we showed that all four pyrrole nitrogens are protonated in both Pr and Pfr states that comprise a ZZZssa and ZZEssa conformation, respectively. Far UV CD data reveal an increase of the α-helical content of at least 3% in the Pr → Pfr transition which goes along with an increased hydrodynamic diameter of Pfr.
We solved the crystal structure of SynCph2(1-2) in the Pr conformation at a resolution of 2.6 Å. The protein crystallizes as an antiparallel dimer, whereby the GAF1 domain connects to GAF2 via a long α-helical linker. The GAF2 domain mimics the overall organization of the PHY domain in protruding a tongue-like extension that covers the chromophore binding pocket of GAF1. The PCB chromophore in the ZZZssa conformation is non-planar and exhibits a high tilt between the B- and C- as well as C- and D-rings. Based on the structural data we performed a mutagenesis study involving the D-ring environment, the propionate interactions of the chromophore and the tongue region. We showed that the B-ring propionate interactions differ in SynCph2(1-2) compared to canonical phytochromes and seem to vary depending on the type of effector domain. Based on the data for the amino acids in the conserved tongue motifs, we suggested a model for the structural changes during photoconversion. Here, a conformational switching of the tongue region is supposed via swap of the tryptophans from the W(G/A)G and WxE motifs that act as anchors in the Pfr structure. Upon photoconversion the Asp-Arg salt bridge between GAF1 and the tongue is broken, the PRxSF motif is reoriented and maybe structurally reorganized to an α-helical structure establishing a new Asp-Ser interaction.
We analyzed the photocycle of SynCph2(1-2) that comprises four and three intermediates in the Pr → Pfr and Pfr → Pr photoconversion, respectively. Ser-385 of the PRxSF and Trp-389 of the WxE motif in the tongue region are ~13 Å and ~15 Å distant from the chromophore’s D-ring, respectively, but they affect the formation of the Pfr state as S385A and W389A variants comprise an altered spectrum after red light illumination. The S385A mutation affects the last intermediate of the Pr → Pfr photoconversion and the first of the back reaction. The W389A mutation already affects the third intermediate that directly decays to a degenerated red light adapted state. The three intermediates of the back reaction to Pr all differ from SynCph2(1-2). In contrast, a phenylalanine substitution of Trp-389 results in wild type behavior. Based on these results we concluded that during the Pr → Pfr photoconversion only the chromophore and its nearest surroundings are altered in the first two intermediates. During formation of intermediate R2 conformational changes occur in the tongue region, involving the tryptophan-switch. Formation of the last intermediate triggers the movement of Ser-385 and the formation of the hydrogen bond network involving the aspartate of GAF1 and Ser-385.
In a work performed with P. Savakis, S. De Causmaecker and V. Angerer and in cooperation with the group of A. Wilde we illuminated the role of the C-terminal GAF3-GGDEF2 module. SynCph2(5-6) comprises a photoconversion between a blue (Pb) and green light absorbing (Pg) state. It covalently attaches PCB to Cys-1022 and to Cys-994 and is able to isomerize the chromophore to PVB. The module can produce c-di-GMP in the GGDEF2 domain in a light dependent manner with Pg as the signaling state. In vivo studies in Synechocystis sp. confirmed these observations. Under blue light conditions the c-di-GMP production of SynCph2(5-6) leads to an inhibition of phototaxis. Nevertheless, only a coproduction of SynCph2(5-6) with SynCph2(1-4) can restore the wild type phenotype under white light thus proving the enzymatic activity of SynCph2(1-4) that is mediated by the EAL domain in degrading c-di-GMP.
Overall, the here presented studies on SynCph2 show the interplay between the photochemical and structural properties in the Pr / Pfr and Pb / Pg interconverting modules as well as their implications in vivo. Future studies will benefit from the enhanced knowledge about intramolecular signaling in phytochromes including the tryptophan switch hypothesis.