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 Microcoleus sp. PCC 7113. 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.

Keywords: adenylate cyclase (adenylyl cyclase); biliprotein; biliverdin; cAMP; cyanobacteria; linear tetrapyrrole; optogenetics; photoreceptor; photoswitch; phototransduction; signal transduction.

Figure 1.

cPAC is a cyanobacterial photoactivated adenylyl cyclase. Shown are domain architectures of cPAC compared with those of other characterized PACs.

Figure 2.

cPAC is blue/green photoswitchable adenylyl cyclase. A, spectra of cPAC displays the distinct B-absorbing (15Z) photostate and the G-absorbing (15E) photostate. B, time course assay of production of cAMP by the G-absorbing 15E state of cPAC with or without calcium present in the media. C, initial rate kinetics of cPAC in the 15E and 15Z photostates. Data are fit to a second order fit derived from the Michaelis-Menten equation: V_o = (V_max[S]²)/(k² + 2k[S] + [S]²). D, HPLC-SEC trace of full-length cPAC in the 15E and 15Z states. The major peak elutes at 19.6 min.

Figure 3.

REC domain of cPAC is essential for optimal activity. A, bar graph of enzyme activity of full-length cPAC and subsequent domain truncations. ΔR3 and ΔR2 denote truncation of the REC domain leaving 3 or 2 helices of the N-terminal GAF domain, respectively, ΔRG denotes the construct of the PAS–cyclase domain and ΔRGP denotes only the cyclase domain (see text for details). B, bar graph of enzyme activity comparing WT cPAC to cPAC(D60E) and cPAC(D60A) mutations. C, schematic of optimal activity conditions for cPAC (B light triggering the 15E-P_g state, phosphorylation of the REC domain in the presence of 5 mm Ca²⁺) as compared with conditions for minimal activity (G light triggering the 15Z-P_b state, with buffer lacking Ca²⁺).

Figure 4.

cPAC can modulate cAMP levels in vivo. A, schematic of cPAC mediating lacZ gene expression via modulation of cAMP levels in (cya⁻) E. coli cells. B, LB (X-Gal/IPTG) plate displaying β-gal cleavage of X-Gal in E. coli expressing cPAC in the section of plate exposed to Bc, whereas Gc vastly reduced X-Gal cleavage in cells. C, in vivo levels of cAMP in E. coli liquid suspension cultures expressing cPAC and incubated under Bc or Gc. cAMP levels were normalized in each culture by O.D. (600 nm) value.

Figure 5.

cPAC as a platform for multicolored fusion constructs. A, jellybean diagram of cPAC fusion constructs. B, alignment showing the N- and C-terminal fusion breakpoints for cPAC and GAF domains of JSC1_58120g3 and RcaE. Yellow highlight denotes residues present in the resultant fusion construct. C, bar graph of enzyme activity of fusion constructs in the 15E and 15Z photostates. D, LB plate (as described above) showing greater X-Gal cleavage and blue coloring in E. coli cells expressing cPAC(RcaE) that are exposed to Gc, as compared with cells exposed to Rc.