The structure of a complete phytochrome sensory module in the Pr ground state

Abstract

Phytochromes are red/far-red photochromic biliprotein photoreceptors, which in plants regulate seed germination, stem extension, flowering time, and many other light effects. However, the structure/functional basis of the phytochrome photoswitch is still unclear. Here, we report the ground state structure of the complete sensory module of Cph1 phytochrome from the cyanobacterium Synechocystis 6803. Although the phycocyanobilin (PCB) chromophore is attached to Cys-259 as expected, paralleling the situation in plant phytochromes but contrasting to that in bacteriophytochromes, the ZZZssa conformation does not correspond to that expected from Raman spectroscopy. We show that the PHY domain, previously considered unique to phytochromes, is structurally a member of the GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) family. Indeed, the tandem-GAF dumbbell revealed for phytochrome sensory modules is remarkably similar to the regulatory domains of cyclic nucleotide (cNMP) phosphodiesterases and adenylyl cyclases. A unique feature of the phytochrome structure is a long, tongue-like protrusion from the PHY domain that seals the chromophore pocket and stabilizes the photoactivated far-red-absorbing state (Pfr). The tongue carries a conserved PRxSF motif, from which an arginine finger points into the chromophore pocket close to ring D forming a salt bridge with a conserved aspartate residue. The structure that we present provides a framework for light-driven signal transmission in phytochromes.

Fig. 1.

Structure and spectral characteristics of the Cph1 phytochrome sensory module from Synechocystis 6803. (A) Domain boundaries of Cph1 phytochrome. In the recombinant Cph1 sensory module described, the C-terminal histidine kinase transmitter (Leu-515–Asn-748) is replaced by a (His)₆ tag. (B) Ribbon representation of the sensory module structure showing the N-terminal α-helix (green) and PAS (blue), GAF (orange) and PHY (red) domains. The PCB chromophore (cyan) is covalently attached to Cys-259. Disordered loop regions (Gln-73–Arg-80, Gly-100–Asp-101, Arg-148–Gln-150, and Glu-463–Gly-465) are indicated as dotted lines. The molecular surface calculated by PYMOL (probe radius, 1.4 Å) is shown in gray. (C) Omit electron density of the adduct between the PCB chromophore and Cys-259 contoured at 2σ. (D) UV/Vis spectra of the Cph1 sensory module in solution at room temperature (red line) and in crystalline form at 100 K (■) in the Pr state (Upper) and after red light irradiation (Lower). Whereas in solution a photoequilibrium at 70% Pfr is reached, the mole fraction is ≈50% in the crystal. Spectra from crystals were recorded at the Cryobench of the ESRF, Grenoble. Photoconversion was done by irradiating for 10 s at room temperature with a 635 nm argon laser focused to 100 μm.

Fig. 2.

Structural comparisons of sensory module domains. (A) Tandem-GAF domain arrangements as observed in Cph1 (orange/red), a murine phosphodiesterase (green) (22), and a cyanobacterial adenylyl cyclase (blue) (24). (B) The PAS/GAF bidomain. The PCB chromophore (cyan), N-terminal α-helix (green) and PAS (light blue) and GAF (orange) domains of Cph1 are superimposed on corresponding elements of the bacteriophytochrome bidomains of D. radiodurans (12) (gray, r.m.s.d. 1.08 Å for 235 C_α-positions) and Rhodopseudomonas palustris (13) (purple, r.m.s.d. 1.10 Å for 252 C_α), which bind biliverdin IXα (BV). (C) Superposition of the Cph1 PHY (red) and GAF (orange) domains and the N-terminal GAF domain of phosphodiesterase 2a (green) (22). Superposition of PHY and its closest homolog, the Cph1 GAF domain, gives an r.m.s.d. of 1.77 Å for 94 equivalent C_α positions. The green asterisk marks the loop of the GAF domain, through which the N terminus passes to form the knot. The cNMPs (green) and the PCB chromophore (cyan) bound within the respective GAF domains are shown with their molecular surfaces, thus showing the partial overlap of their binding sites.

Fig. 3.

The tongue and the chromophore binding pocket. (A) Space-filling model of Cph1 (Left) in comparison with known bacteriophytochrome structures (12, 13). The PCB chromophore (cyan) is completely sealed from solvent access by the tongue (dark red) in contrast to the exposed biliverdin (green) in the incomplete bidomains. (B and C) The tripartite PCB-binding pocket of Cph1 comprising the GAF-domain (orange), the tongue-like protrusion from the PHY domain (red) and the N-terminal α₁-helix (green). Waters are shown as red spheres. (B) Edge-on view of the pocket highlighting the collinear arrangement of the N-terminal α₁-helix and α₇-helix of the GAF domain and their interaction with the chromophore and the tongue. (C) The conformation of the PCB chromophore (cyan) within the PCB-binding site adopts a ZZZssa configuration similar to that of BV in bacteriophytochromes (12, 13). For clarity, α₈-helix of the GAF domain as well as Tyr-263 and Phe-475 have been omitted.

Fig. 4.

Comparisons of the chromophore pocket in Cph1 (orange) and bacteriophytochrome (yellow) (12) with their respective chromophores PCB (cyan) and biliverdin (green). Waters are shown as red spheres. (A) The subsite for interactions between the bilin propionate groups and the GAF domain, showing the different conformations adopted by Arg-222 and Phe/Tyr-216 in Cph1 and bacteriophytochrome. (B and C) The ring D microenvironment in Cph1 and bacteriophytochrome, respectively. The molecular surfaces of the proteins (gray) show similar cavities with a triangular cross section providing space for the Z→E photoflip. The chromophore is sealed off from the solvent in the case of the Cph1 complete sensory module, whereas the bacteriophytochrome bidomain pocket is open to the solvent (note the numerous waters).