Crystallographic and electron microscopic analyses of a bacterial phytochrome reveal local and global rearrangements during photoconversion

Phytochromes are multidomain photoswitches that drive light perception in plants and microorganisms by coupling photo-reversible isomerization of their bilin chromophore to various signaling cascades. How changes in bilin conformation affect output by these photoreceptors remains poorly resolved and might include several species-specific routes. Here, we present detailed three-dimensional models of the photosensing module and a picture of an entire dimeric photoreceptor through structural analysis of the Deinococcus radiodurans phytochrome BphP assembled with biliverdin (BV). A 1.16-Å resolution crystal structure of the bilin-binding pocket in the dark-adapted red light-absorbing state illuminated the intricate network of bilin/ protein/water interactions and confirmed the protonation and ZZZssa conformation of BV. Structural and spectroscopic comparisons with the photochemically compromised D207A mutant revealed that substitutions of Asp-207 allow inclusion of cyclic porphyrins in addition to BV. A crystal structure of the entire photosensing module showed a head-to-head, twisted dimeric arrangement with bowed helical spines and a hairpin protrusion connecting the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) and phytochrome-specific (PHY) domains. Akey conserved hairpin feature is its anti-parallel, two β-strand stem, which we show by mutagenesis to be critical for BphP photochemistry. Comparisons of single particle electron microscopic images of the full-length BphP dimer in the red lightabsorbing state and the photoactivated far-red light-absorbing state revealed a large scale reorientation of the PHY domain relative to the GAF domain, which alters the position of the downstream histidine kinase output module. Together, our data support a toggle model whereby bilin photoisomerization alters GAF/PHY domain interactions through conformational modification of the hairpin, which regulates signaling by impacting the relationship between sister output modules.