On the Role of the Conserved Histidine at the Chromophore Isomerization Site in Phytochromes.

Phytochromes are sensory photoreceptors that use light to drive protein structural changes, which in turn trigger physiological reaction cascades. The process starts with a double-bond photoisomerization of the linear methine-bridged tetrapyrrole chromophore in the photosensory core module. The molecular mechanism of the photoconversion depends on the structural and electrostatic properties of the chromophore environment, which are highly conserved in related phytochromes. However, the specific role of individual amino acids is yet not clear. A histidine in the vicinity of the isomerization site is highly conserved and almost invariant among all phytochromes. The present study aimed at analyzing its role by taking advantage of a myxobacterial phytochrome SaBphP1 from Stigmatella aurantiaca, where this histidine is naturally substituted with a threonine (Thr289), and comparing it to its normal, His-containing counterpart from the same organism SaBphP2 (His275). We have carried out a detailed resonance Raman and IR spectroscopic investigation of the wild-type proteins and their respective His- or Thr-substituted variants (SaBphP1-T289H and SaBphP2-H275T) using the well-characterized prototypical phytochrome Agp1 from Agrobacterium fabrum as a reference. The overall mechanism of the photoconversion is insensitive toward the His substitution. However, the chromophore geometry at the isomerization site appears to be affected, with a slightly stronger twist of ring D in the presence of Thr, which is sufficient to cause different light absorption properties in SaBphP1 and SaBphP2. Furthermore, the presence of His allows for multiple hydrogen-bonding interactions with the ring D carbonyl which may be the origin for the geometric differences of the C-D methine bridge compared to the Thr-containing variants. Other structural and mechanistic differences are independent of the presence of His. The most striking finding is the protonation of the ring C propionate in the Pfr states of SaBphP2, which is common among bathy phytochromes but so far has not been reported in prototypical phytochromes.

High-resolution crystal structures of transient intermediates in the phytochrome photocycle.

Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting Pr state and the light-activated Pfr state. Light signals are transduced as structural change through the entire protein to modulate its activity. It is unknown how the Pr-to-Pfr interconversion occurs, as the structure of intermediates remains notoriously elusive. Here, we present short-lived crystal structures of the photosensory core modules of the bacteriophytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray free electron laser 5 ns and 33 ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway that extends through the entire protein. The snapshots show with atomic precision how the signal progresses from the chromophore, explaining how plants, bacteria, and fungi sense red light.

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

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.