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

Abstract

Sensing of red and far-red light by bacteriophytochromes involves intricate interactions between their bilin chromophore and the protein environment. The light-triggered rearrangements of the cofactor configuration and eventually the protein conformation enable bacteriophytochromes to interact with various protein effector domains for biological modulation of diverse physiological functions. Excitation of the holoproteins by red or far-red light promotes the photoconversion to their far-red light–absorbing Pfr state or the red light-absorbing Pr state, respectively. Because prototypical bacteriophytochromes have a parallel dimer architecture, it is generally assumed that symmetric activation with two Pfr state protomers constitutes the signaling-active species. However, the bacteriophytochrome from Idiomarina species A28L (IsPadC) has recently been reported to enable long-range signal transduction also in asymmetric dimers containing only one Pfr protomer. By combining crystallography, hydrogen–deuterium exchange coupled to MS, and vibrational spectroscopy, we show here that Pfr of IsPadC is in equilibrium with an intermediate “Pfr-like” state that combines features of Pfr and Meta-R states observed in other bacteriophytochromes. We also show that structural rearrangements in the N-terminal segment (NTS) can stabilize this Pfr-like state and that the PHY-tongue conformation of IsPadC is partially uncoupled from the initial changes in the NTS. This uncoupling enables structural asymmetry of the overall homodimeric assembly and allows signal transduction to the covalently linked physiological diguanylate cyclase output module in which asymmetry might play a role in the enzyme-catalyzed reaction. The functional differences to other phytochrome systems identified here highlight opportunities for using additional red-light sensors in artificial sensor–effector systems.

Intramolecular Proton Transfer Controls Protein Structural Changes in Phytochrome

“…This implies that a part of the structural changes of the chromophore and its immediate environment occurs independent of protein structural changes. In this context, it is interesting to note that uncoupling of chromophore and protein conformational transitions has also been reported for prototypical bacteriophytochrome. − …”

Improved fluorescent phytochromes for in situ imaging

“…Here again, despite the rather similar 3D structures of Cph1 and Dr BphP, homologous mutations have quite different effects. Although absence of the PHY domain generally blocks Pfr formation 45 , 47 , exceptions are Gm .phyA 48 , the monomeric mutant of Dr BphP 45 and Is PadC 49 that form Pfr-like states. It might be that the PHY domain tongue contributes to the rigidity of the chromophore pocket, sometimes enhancing and sometimes inhibiting fluorescence, but usually as a requirement for Pfr formation.…”

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…The versatility of HDX-MS has also enabled detailed studies of conformational changes due to light. Light sensing proteins in their resting and activated forms have been studied to provide revolutionary insights into their mechanisms of photoactivation. − For example, Etzl et al were able to discern the changes in the structure of phytochrome-activated guanylate cyclase upon illumination with red light (Figure ). This application is another illustration of the capability of HDX-MS to provide detailed insight into a system that cannot be analyzed by other means.…”