Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain

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.

Molecular basis for GTP recognition by light‐activated guanylate cyclase RhGC

“…1B and [20]). In many of our crystallization conditions, we obtained crystals with unit cells comprising monomeric or unconventional head-tohead disulphide bond-stabilized dimeric assemblies that have been reported by others [11,20]; these will not be discussed further.…”

“…Mechanistic insights into the activity of class III NCs have been derived from dozens of crystal structures of AC domains in a ligand-free form or bound to substrate-based inhibitors, substrates or ATP analogues in various conformations [3][4][5][6][7][8][9][10][11]. Structural analysis suggests that the AC dimer undergoes a functional conformational change that involves 'closing' of the active site region that brings the catalytic residues distributed across the subunit interface into the proper register.…”

“…Most of the GC structures available to date represent ligand-free, atypical head-to-head or canonical head-to-tail dimers that are inactive due to misaligned active site residues [15][16][17][18][19][20]. A few reports describe crystal structures of the GC domain in the active conformation; however, they exploit enzymes of altered specificity [10,11]. Important new information has recently emerged from the 3.8 A resolution cryo-EM study of the human soluble GC (sGC) in its inactive and GMPCPP-bound NO-activated state [21], suggesting that all type III NCs undergo dynamic structural changes as part of the catalytic cycle.…”

“…Crystal structures of the soluble GC domain of B. emersonii RhGC (BeGC) [20] and a calcium-and ATPaS-bound dimeric form of the C. anguillulae RhGC GC domain with several mutations that transform substrate specificity into an AC (CaACÁATPaSÁCa 2+ ) [11] have been recently described. Here, we report the crystal structure of the wild-type GC domain of RhGC from C. anguillulae in its GTPand calcium-bound form (CaGCÁGTPÁCa 2+ ).…”

Structural insights into the mechanism of rhodopsin phosphodiesterase

“…Although HeRs share low sequence identity with the other rhodopsins and adopt the inverted membrane topology, the crystal structures revealed that HeR comprises 7TMs with the retinal chromophore, as in the other rhodopsins [39][40][41] . Unlike these rhodopsins, the enzyme rhodopsins have long N-termini, and are predicted to have 1-2 additional transmembrane helices 11,12,42 , while the existence of TM0 and its function still have remained to be elucidated. The current Rh-PDE structures revealed that TM0 certainly exists and plays key roles in the stable retinal binding to TM1-7 and photoactivation ( Fig.…”