Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC

Abstract

RhoGC is a fusion protein from the aquatic fungus , combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from with (GCwCC) and without (GC) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GC have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2',3'-dideoxyguanosine-5'-triphosphate, MnCl, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GC monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.

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

“…Although RhGC is anticipated to act as a dimer like all other GCs [29,30], the isolated GC domain is monomeric in solution ( Fig. 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.…”

“…In particular, the current literature does not provide a sufficient explanation for the NC substrate selectivity. 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].…”

“…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+ ).…”

Structure/function of the soluble guanylyl cyclase catalytic domain

“…2). Several structures of apo inactive cyclase domains from bacteria, algae, fungus, and human have been solved, but a structure of the holo active form remains elusive [40–44]. Several groups have reported a high propensity for ββGC cat homodimers to form both in solution and during crystallization attempts [42,43].…”

“…Very recently, the structure of the catalytic guanylyl cyclase domain of the fusion protein RhoGC from the aquatic fungus Blastocladiella emersonii was solved [44]. The homodimeric transmembrane protein senses light through a rhodopsin domain (residues 176-388) and transduces the signal via a coiled-coil domain to the cytosolic catalytic GC Rho domain (residues 443-626) where GTP can be cyclized for phototaxis [64,65].…”