Sugiura, M. | Search bioRxiv

bioRxiv2020

Structural insights into the mechanism of rhodopsin phosphodiesterase

Rhodopsin 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 [A] 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 [A]) 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.

biophysics↗

bioRxiv2019

High-throughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations

Insects voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein are known to confer insecticide resistance and are selected under insecticide pressure. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure of gene VGSC, it is usually challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes via annotated CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all homologous VGSC CDSs except tiny exons from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency by virtue of the high conservation of VGSC at the nucleotide level. Furthermore, we developed an automated bioinformatic analysis pipeline to genotype VGSC after capture sequencing--MoNaS (Mosquito Na+ channel mutation Search)--which conducts mapping of reads, variant calling, and variant annotation for nonsynonymous mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetics studies on resistance alleles with respect to the origin, selection, and migration of both clinically and agriculturally important insect pests.

genetics↗