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.
Highlights
IntroductionCrystals were harvested under low-intensity nonactinic green-light conditions (520 Ϯ 20 nm LED) by transferring the crystals to a cryoprotectant solution (reservoir solution containing 10% glycerol) and subsequent flash-freezing in liquid nitrogen
Crystals were harvested under low-intensity nonactinic green-light conditions (520 Ϯ 20 nm LED) by transferring the crystals to a cryoprotectant solution and subsequent flash-freezing in liquid nitrogen
RR analysis indicates very similar 15E protonated BV environments in both the full-length IsPadC and in the IsPadCPG dimerE/E that lacks a disordered/␣-helical PHYtongue. These findings show that the observed structural rearrangements in the NTS precede tongue refolding and, thereby, are sufficient to allow the stabilization of a Pfr-like state in full-length IsPadC
Summary
Crystals were harvested under low-intensity nonactinic green-light conditions (520 Ϯ 20 nm LED) by transferring the crystals to a cryoprotectant solution (reservoir solution containing 10% glycerol) and subsequent flash-freezing in liquid nitrogen. Diffraction data for IsPadCPG monomerZ were collected at beamline ID23-2 of the European Synchrotron Radiation Facility (ESRF), and IsPadCPG dimerE/E diffraction data were collected at beamline P11 of the Deutsches ElektronenSynchrotron (DESY). Data were processed using the XDS program package [50]
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