Abstract
The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsin-guanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio >1000. After light excitation the putative signaling state forms with τ = 31 ms and decays with τ = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 Å) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.
Highlights
The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses
We further characterize the RhGC from the fungus Catenaria anguillulae (CaRhGC), which is another member of the chitin-walled Blastocladiomycota[11,12], and the adenylyl cyclase CaRhAC resulting from the point mutations E497K, C566D in Xenopus oocytes and hippocampal neurons
We present the crystal structure of the ligand-bound adenylyl cyclase domain (CaAC) at 2.25 Å resolution, which reveals the mechanistic basis for the change from cGMP to cAMP production
Summary
The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. We present the characterization of the rhodopsinguanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio >1000. The crystal structure of the ligand-bound AC domain (2.25 Å) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light. YFP-CaRhGC together with the YFPCaRhAC mutant, expand the optogenetic toolbox allowing control of cAMP/cGMP signaling within milliseconds close to cellular membranes
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