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

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 PRSF 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 (Tyr) in the phytochrome from the extremophile bacterium 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 Tyr 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 Tyr 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.

Distinct chromophore–protein environments enable asymmetric activation of a bacteriophytochrome-activated diguanylate cyclase

“…This interpretation is in line with previous findings on DrBphP, which suggested that the tongue transition constitutes a thermal equilibrium that is affected by but not strictly linked to the chromophore state (42). The degree of tongue coupling with the cofactor apparently not only influences the thermal back reaction to Pr (see below) but also the efficiency of the photochemical reaction.…”

“…Apparently, there are system-specific differences in the photocycles of members of the phytochrome superfamily, frequently brought about by a protonation-depen-dent heterogeneity of different parent or intermediate states, in line with previous results (36 -40). Eventually, this heterogeneity influences structural changes involved in signal propagation (41) and, together with other properties of the bilin cofactor, might be influenced by the degree of structural coupling with functional elements affecting the BV environment (7,42) and hence the stability of specific cofactor configurations, conformations, and/or protonation states.…”

Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

“…Phytochrome function includes several structural tiers that range from the chromophore surroundings to large-scale structural changes in the entire protein. These tiers are in dynamic equilibrium, which can be shifted by the other tiers and by external factors 68 . The level of phytochrome output activity can be considered to be in an equilibrium between histidine kinase and phosphatase activities 18,61 ( Figure 6B).…”

Signaling mechanism of phytochromes in solution

“…Another theory is that the knot limits the flexibility of the protein in order to prevent undesirable loss of entropy due to large domain movements upon photoisomerization of the chromophore. 7 However, as current crystal structures doe not reveal any changes in the knot region between Pr and Pfr, 15,17,18,[20][21][22][23] these ideas remain speculative.…”

“…How can that this be rationalized? It is known that the photophysical state of the chromophore can be uncoupled from the conformation of the phytochrome in crystal structures 15,22. Crystal packing can alter protein conformations, with packing energies on the order of tertiary or quarternary interactions.…”