On the (un)coupling of the chromophore, tongue interactions, and overall conformation in a bacterial phytochrome

Heikki Takala,Heli K Lehtivuori,Oskar Berntsson,Ashley Hughes,Rahul Nanekar,Stephan Niebling,Matthijs Panman,Léocadie Henry,Andreas Menzel,Sebastian Westenhoff,Janne A Ihalainen

doi:10.1074/jbc.ra118.001794

Abstract

Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Their light responses are thought to follow a conserved structural mechanism that is triggered by isomerization of the chromophore. Downstream structural changes involve refolding of the so-called tongue extension of the phytochrome-specific GAF-related (PHY) domain of the photoreceptor. The tongue is connected to the chromophore by conserved DIP and PRXSF motifs and a conserved tyrosine, but the role of these residues in signal transduction is not clear. Here, we examine the tongue interactions and their interplay with the chromophore by substituting the conserved tyrosine (Tyr263) in the phytochrome from the extremophile bacterium Deinococcus radiodurans with phenylalanine. Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD-PHY fragments, we show that the absence of the Tyr263 hydroxyl destabilizes the β-sheet conformation of the tongue. This allowed the phytochrome to adopt an α-helical tongue conformation regardless of the chromophore state, hence distorting the activity state of the protein. Our crystal structures further revealed that water interactions are missing in the Y263F mutant, correlating with a decrease of the photoconversion yield and underpinning the functional role of Tyr263 in phytochrome conformational changes. We propose a model in which isomerization of the chromophore, refolding of the tongue, and globular conformational changes are represented as weakly coupled equilibria. The results also suggest that the phytochromes have several redundant signaling routes.

Highlights

Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light– absorbing (Pr) and far-red light–absorbing (Pfr) states
Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD–phytochrome-specific GAF-related (PHY) fragments, we show that the absence of the Tyr263 hydroxyl destabilizes the ␤-sheet conformation of the tongue
Phytochromes are a family of red/far-red light–sensing photoreceptors found in plants, fungi, cyanobacteria, and eubacteria

Summary

The abbreviations used are

Pr, red light–absorbing; Pfr, far-red light–absorbing; CBD, chromophore-binding domain; PHY, phytochrome-specific GAF-related; PSM, photosensory module; PDB, Protein Data Bank; DrBphP, D. radiodurans bacteriophytochrome; PAS, Per/Arnt/Sim; GAF, cGMP phosphodiesterase, adenylyl cyclase, FhlA; ESRF, European Synchrotron Radiation Facility; RMSD, root mean square deviation. By a Y263F mutation, we pinpoint the multiple roles that this tyrosine has for the photocycle and for stabilizing the tongue interactions. Mophore are important for the initial photoreactions These include a conserved DIP motif in the GAF domain [3]. The initial photoreactions in the immediate chromophore surroundings lead to changes in a so-called tongue extension of the PHY domain. The tongue contains a conserved PRXSF motif that binds the DIP motif of the GAF domain. In the Pr state, a conserved Asp residue of the DIP motif, Asp207 in the Deinococcus radiodurans phytochrome (DrBphP), forms a salt bridge with an Arg residue (Arg466) of the tongue [3, 9, 11]. The important DIP and PRXSF motifs are closely associated with a conserved tyrosine (Tyr263).

Results

Discussion

Experimental procedures