Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells

Abstract

Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in nonphotosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium sp. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibits a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for the design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells, tissues, and whole organisms with light across the visible spectrum and into the near IR.

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

“…Unlike canonical phytochromes, CBCR photosensory modules consist of one or more stand-alone GAF domains that are sufficient for covalent attachment of bilin and photoconversion. These small CBCR domains have also been used as light-sensing modules in a variety of synthetic biology applications (27)(28)(29)(30)(31)(32). In contrast to the typical red/far-red phytochromes, CBCRs are able to sense all colors of light from near UV to far-red utilizing a common phycocyanobilin (PCB) chromophore precursor (22)(23)(24)26).…”

The Red‐/Green‐Switching GAF3 of Cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803 Regulates the Activity of an Adenylyl Cyclase

“…RGS showed a rapid red/green response and was easily controlled by monochromatic light, considering the wide separation of the absorption maxima between both the red‐light‐absorbing thermostable state (P r ) and the green‐light‐absorbing metastable state (P g ). A new characterized CBCR could act as an optogenetics tool not only between blue‐ and green‐light‐induced states, but also near‐infrared‐induced states . It turned out that an optimized linker was needed to control downstream enzyme activity, which is key for designing optogenetics tools .…”

“…Photoreceptors of other classes and functions have already been employed as optogenetics tools, such as the flavin‐binding blue‐light‐sensing using FAD (BLUF) domain receptors; the light, oxygen, voltage (LOV) domain proteins; the photosensory PAS‐GAF‐PHY domain from (bacterio)phytochromes; and also the CBCR that is reversibly switched by blue and green light . It was demonstrated that the photosensory N‐terminal chromophore‐binding domain could successfully control a C‐terminal transduction domain, either its inherent functional domain or an unnaturally existing combination of sensing and signaling domains after their combination through an artificial fusion peptide.…”

Green, orange, red, and far-red optogenetic tools derived from cyanobacteriochromes

“…As a target we chose Am1_c0023g2, a red/green GAF domain from Acaryochloris marina, that can bind two distinct bilins -PCB or biliverdin (BV) (27 Am1_c0023g2 can be made responsive to either green, orange, red or far-red light. Optogenetic tools that respond to green light are currently limited to complex cobalamin based systems (28,29) and CcaS/CcaR or cPAC systems that have predefined functions -gene expression (30), or adenylyl cyclase activity respectively (26). In addition, small, monomeric optogenetic tools that operate with far-red light are useful since far-red absorption provides deep tissue penetration ability for use in living animals (31).…”