Novel Modular Rhodopsins from Green Algae Hold Great Potential for Cellular Optogenetic Modulation Across the Biological Model Systems

Abstract

Light-gated ion channel and ion pump rhodopsins are widely used as optogenetic tools and these can control the electrically excitable cells as (1) they are a single-component system i.e., their light sensing and ion-conducting functions are encoded by the 7-transmembrane domains and, (2) they show fast kinetics with small dark-thermal recovery time. In cellular signaling, a signal receptor, modulator, and the effector components are involved in attaining synchronous regulation of signaling. Optical modulation of the multicomponent network requires either receptor to effector encoded in a single ORF or direct modulation of the effector domain through bypassing all upstream players. Recently discovered modular rhodopsins like rhodopsin guanylate cyclase (RhoGC) and rhodopsin phosphodiesterase (RhoPDE) paves the way to establish a proof of concept for utilization of complex rhodopsin (modular rhodopsin) for optogenetic applications. Light sensor coupled modular system could be expressed in any cell type and hence holds great potential in the advancement of optogenetics 2.0 which would enable manipulating the entire relevant cell signaling system. Here, we had identified 50 novel modular rhodopsins with variant domains and their diverse cognate signaling cascades encoded in a single ORF, which are associated with specialized functions in the cells. These novel modular algal rhodopsins have been characterized based on their sequence and structural homology with previously reported rhodopsins. The presented novel modular rhodopsins with various effector domains leverage the potential to expand the optogenetic tool kit to regulate various cellular signaling pathways across the diverse biological model systems.

Keywords: channelrhodopsins; cyclase; enzyme-rhodopsin; optogenetics; phosphodiesterase; two-component system.

Figure 1

Schematic representation of domains present in modular microbial type rhodopsins. The schematic representation shows rhodopsin with modular domain(s), the black line represents full-length protein, and domains are depicted by geometric structures (Figure not to scale). (A) Domain organization of modular Channelrhodopsins (ChRs). ChR coupled with FimV (peptidoglycan binding protein), MED15 (mediator of RNA polymerase transcription factor subunit 15), and UL36 (large tegument protein) were found in three different algae. (B) Rhodopsin coupled HisK and RR form the largest group of modular domains and others have additional unique effector domains like cyclase (Cyc), sterile alpha subunit (SAM), structural maintenance of chromosome_N-terminus (SMC_N), transposase (Tnp2), major viral transcription factor ICP4 homolog (ICP4), 104kDa microneme/rhoptry (Mn 104) and bacterial flagellar motor protein (MotB). (C) Modular rhodopsin with rhodopsin preceded by unique domain at N-terminus; ATP-dependent 26S proteasome (RPT1) and bromodomain (BRD) in GpRh5 and tricopeptide (TPR) in OtRh2. (D) Modular rhodopsin lacking HisK and RR; GtRh1 possesses Spore lysis A and Ryanodine receptor (SPRY) domain that regulates innate and adaptive immune response and domain of unknown function (DUF), GtRh2 and 3 possess MED15. AsRh1 possesses regulator of V-ATPase of vacuolar membrane protein 1 (Rav1) and WD40 at N-terminus.

Figure 2

Comparison of novel channelrhodopsins and mapping of the important amino acid residues: Modular ChRs (KnRh3, TsRh1, and GpRh1) were aligned with other ChRs (ChR1 and ChR2 Figure 1. & VChR2 from V. carteri, MvChR1 from M. viride. Helices 1–7 are depicted by a black bar and marked in roman numbers. Retinal binding lysine is marked by the red arrow; proton acceptor/donor and cysteine hydrogen-bonded to proton donor (DC pair) are marked by the pink arrow; arginine is important for primary translocation of the proton is marked by an orange arrow.

Figure 3

Comparison of light sensor domain of the modular rhodopsin among different algae: Most conserved third, fourth, sixth, and seventh helices of rhodopsin are depicted here. Numbering was adapted according to the protein of BR. 1KGB: Bacteriorhodopsin, 1UAZ: Archaerhodopsin-1, 1VGO: Archaerhodopsin-2, 1El2: Halorhodopsin, 1H2S: Sensory Rhodopsin II, 1XIO: Anabaena sensory rhodopsin.

Figure 4

Sequence relatedness of the microbial type modular rhodopsin: Rhodopsin domain phyletic topology shows clustering of typical MTR and extended C-terminus rhodopsins in a separate clade. Modular rhodopsins formed a different clade. KnRh3, GpRh1 and TsRh1 grouped with ChRs. AsRh4 with Rav1 domain is the only modular rhodopsin grouped with proton pumping algal rhodopsin CsR (Rhodopsin from Coccomyxa subllipsodea). GtRh1 was unique and separated from all lying between BR and HR.

Figure 5

Multiple sequence alignment of the cyclase domain of modular rhodopsins: Cyclase domains of modular rhodopsins were aligned with canonical cyclase proteins. Black arrowhead depicts metal-binding residue, purple arrowhead shows substrate-binding residue and the red arrowhead shows transition state stabilizing the residues of the cyclases.