Structural insights into the mechanism of rhodopsin phosphodiesterase

Abstract

AbstractRhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase (Rh-GC) and photosensitive adenylyl cyclase (PAC). Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The 2.6 Å resolution crystal structure of the transmembrane domain revealed a new topology of rhodopsin, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the transmembrane and PDE domain (2.1 Å) with their connecting linkers. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.