Abstract
Phytochromes are multidomain photoswitches that drive light perception in plants and microorganisms by coupling photoreversible 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. A key 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 light-absorbing 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.
Highlights
Phytochromes are dimeric bili-proteins central to photoperception by plants and microorganisms
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
We present here a collection of structural images for the bacterial Phy BphP from Deinococcus radiodurans with a canonical PAS-GAF-PHY domain photosensing module (PSM) followed by a histidine kinase (HK) domain output module (OPM) [35]
Summary
Phytochromes are dimeric bili-proteins central to photoperception by plants and microorganisms. 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. The phytochrome (Phy) superfamily encompasses a structurally diverse collection of photochromic photoreceptors in plants and microorganisms that use a bilin (or open-chain tetrapyrrole) chromophore for light detection [1,2,3]. They typically exist in two forms, a dark-adapted red light-absorbing state (Pr) that is biologically inactive and a metastable far-red light-absorbing state (Pfr) that is biologically active. In canonical Phys, the PSM sequentially contains a Period/Arnt/Single-minded (PAS) domain of unknown function, a signature cGMP phosphodiesterase/ adenylyl cyclase/FhlA (GAF) domain that cradles the bilin
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