High-resolution crystal structures of a myxobacterial phytochrome at cryo and room temperatures

Abstract

Phytochromes (PHYs) are photoreceptor proteins first discovered in plants, where they control a variety of photomorphogenesis events. PHYs as photochromic proteins can reversibly switch between two distinct states: a red light (Pr) and a far-red light (Pfr) absorbing form. The discovery of Bacteriophytochromes (BphPs) in nonphotosynthetic bacteria has opened new frontiers in our understanding of the mechanisms by which these natural photoswitches can control single cell development, although the role of BphPs in vivo remains largely unknown. BphPs are dimeric proteins that consist of a photosensory core module (PCM) and an enzymatic domain, often a histidine kinase. The PCM is composed of three domains (PAS, GAF, and PHY). It holds a covalently bound open-chain tetrapyrrole (biliverdin, BV) chromophore. Upon absorption of light, the double bond between BV rings C and D isomerizes and reversibly switches the protein between Pr and Pfr states. We report crystal structures of the wild-type and mutant (His275Thr) forms of the canonical BphP from the nonphotosynthetic myxobacterium Stigmatella aurantiaca (SaBphP2) in the Pr state. Structures were determined at 1.65 Å and 2.2 Å (respectively), the highest resolution of any PCM construct to date. We also report the room temperature wild-type structure of the same protein determined at 2.1 Å at the SPring-8 Angstrom Compact free electron LAser (SACLA), Japan. Our results not only highlight and confirm important amino acids near the chromophore that play a role in Pr-Pfr photoconversion but also describe the signal transduction into the PHY domain which moves across tens of angstroms after the light stimulus.

FIG. 1.

Comparisons of the SaBphP2 PCM (a) in the wild-type and SaBphP1 PCM (b) in the wild-type forms. The PAS, GAF, and PHY domains are colored yellow, green, and magenta, respectively. The PCM is a dimer with one monomer highlighted in gold. The kink at the helical transition from GAF to PHY is apparent in panel (b) and BV is marked in panels (a) and (b). (c) Schematic presentation of domain organization of BphPs is below with sequence numbers provided for SaBphP2.

FIG. 2.

Hydrogen-bonding network of the conserved amino acids and neighboring water molecules stabilizing BV chromophore in the Pr state of SaBphP2 wild-type (a) and SaBphP1 wild-type (b). Hydrogen bonds are marked with dashed lines, and amino acids located in the GAF domain are highlighted in green. (c) Partial protein sequence alignment highlighting conserved amino acids stabilizing propionate side chains of BV chromophore (in cyan) as well as A-D pyrrole rings. (d) Table with conserved amino acids from SaBphP1, SaBphP2, DrBphP, and bathy PaBphP with specific BV interactions as identified in the Pr and Pfr states.

FIG. 3.

PRXSF motif of the PHY domain and its specific hydrogen-bonds with the amino acids of the GAF domain in the Pr state of SaBphP2 wild-type (a) and Pfr states of bathy PaBphP (b) and classical DrBphP (c) phytochrome proteins. (d) Partial protein sequence alignment highlighting critical amino acids in the GAF (green) and PHY (magenta) domains that form hydrogen bond interactions (highlighted with dashed lines). The table summarizes amino acid interactions of GAF and PHY domains of SaBphP2 (Pr), classical DrBphP (Pfr), and bathy PaBphP (Pfr).

FIG. 4.

Structural comparison with important BphPs. (a) Superposition of the SaBphP2 PCM (Pr) (yellow, green, and magenta) onto the full length BphP with a diguanylyl cyclase effector domain from Idiomarina sp. (gray). (b) Superposition of the SaBphP1 PCM (yellow, green, and magenta) on the full length BphP with a diguanylyl cyclase effector domain from Idiomarina sp. (gray). The helical kink is absent in (a) and marked in (b). In both SaBphPs, the sensory tongue displays a β-sheet structure indicative of the Pr state.

FIG. 5.

Two proposed pathways for signal transduction highlighted in the Pr state of SaBphP2 wild-type protein. His 275 together with the conserved Arg 207 and Arg 157 undergoes significant conformational changes upon Pfr formation as observed in the classical DrBphP and the bathy PaBphP [Figs. 3(b) and 3(c)]. The protein backbone connecting to the conserved PASDIP motif and D192 is shown in gray.

FIG. 6.

Photoactivity of SaBphP2 crystals and expected protein structural changes upon red-light illumination. (a) Absorption spectra of the crystals of wild-type SaBphP2 PAS-GAF-PHY before and after light illumination. Black line: spectrum in the dark; blue line: spectrum after 1 min of illumination with a 660 nm light emitting diode at ∼5 mW/mm². Spectral changes are reversible. Red line: difference spectrum (3× enhanced). Dashed red line: zero line. Red arrow: illumination wavelength. (b) Superposition of the SaBphP2 PCM (Pr) (yellow, green, and magenta) onto the DrBphP PCM (Pfr) in gray. The PHY domains are displaced substantially in the Pfr structure.