Rhodopsin-guanylyl Cyclase Research Articles

The Functionality of the DC Pair in a Rhodopsin Guanylyl Cyclase from Catenaria anguillulae

Rhodopsin guanylyl cyclases (RGCs) belong to the class of enzymerhodopsins catalyzing the transition from GTP into the second messenger cGMP, whereas light-regulation of enzyme activity is mediated by a membrane-bound microbial rhodopsin domain, that holds the catalytic center inactive in the dark. Structural determinants for activation of the rhodopsin moiety eventually leading to catalytic activity are largely unknown. Here, we investigate the mechanistic role of the D283-C259 (DC) pair that is hydrogen bonded via a water molecule as a crucial functional motif in the homodimeric C. anguillulae RGC. Based on a structural model of the DC pair in the retinal binding pocket obtained by MD simulation, we analyzed formation and kinetics of early and late photocycle intermediates of the rhodopsin domain wild type and specific DC pair mutants by combined UV–Vis and FTIR spectroscopy at ambient and cryo-temperatures. By assigning specific infrared bands to S-H vibrations of C259 we are able to show that the DC pair residues are tightly coupled. We show that deprotonation of D283 occurs already in the inactive L state as a prerequisite for M state formation, whereas structural changes of C259 occur in the active M state and early cryo-trapped intermediates. We propose a comprehensive molecular model for formation of the M state that activates the catalytic moiety. It involves light induced changes in bond strength and hydrogen bonding of the DC pair residues from the early J state to the active M state and explains the retarding effect of C259 mutants.

A Bioengineered In Vitro Model to Assess AAV-Based Gene Therapies for Cyclic GMP-Related Disorders.

The emergence of efficient viral vectors derived from adeno-associated viruses (AAV) has led many groups to develop gene therapies for inherited monogenic diseases, such as retinal dystrophies. To evaluate the potency of new gene therapy vectors in a preclinical context, it is common to use animal models, such as gene-deficient or mutant animal models of a given human disease, and then assess vision restoration with functional or behavioral assays. While such animal models are invaluable to the preclinical testing process, they cannot be readily used as batch release tests during manufacturing or to validate biological activity at later stages of development. There is therefore a need for rapid and reliable in vitro models that can determine whether therapeutic vectors have delivered their cargo gene, and more importantly, whether this has resulted in the intended biological activity. Given our previous experience, we chose CNGA3-linked achromatopsia to develop a cell-based system to verify biological activity of AAV vectors designed to deliver a healthy CNGA3 gene copy into human cone photoreceptors. Our system is based on an immortalized cell line with high susceptibility to AAV transduction, i.e., HeLa cells, which we engineered to express a fungal rhodopsin guanylyl cyclase (RhGC) from Blastocladiella emersonii and a sensitive genetically encoded calcium indicator (GECI) under the control of a tetracycline operator. Using this system, we were able to confirm and quantify the function of the ion channel encoded by AAV/CNGA3 and differentiate between AAV vector potencies with a simple fluorometric assay. Finally, we show that this approach can be readily adapted for the assessment of phosphodiesterase function.

The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy.

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases, and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here, we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.

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 and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, 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 rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. 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.

Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3\u2009\xc5 structure of the adenylyl cyclase domain

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.

Absorption and Emission Spectroscopic Investigation of Thermal Dynamics and Photo-Dynamics of the Rhodopsin Domain of the Rhodopsin-Guanylyl Cyclase from the Nematophagous Fungus Catenaria anguillulae.

The rhodopsin-guanylyl cyclase from the nematophagous fungus Catenaria anguillulae belongs to a recently discovered class of enzymerhodopsins and may find application as a tool in optogenetics. Here the rhodopsin domain CaRh of the rhodopsin-guanylyl cyclase from Catenaria anguillulae was studied by absorption and emission spectroscopic methods. The absorption cross-section spectrum and excitation wavelength dependent fluorescence quantum distributions of CaRh samples were determined (first absorption band in the green spectral region). The thermal stability of CaRh was studied by long-time attenuation measurements at room temperature (20.5 °C) and refrigerator temperature of 3.5 °C. The apparent melting temperature of CaRh was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 62 ± 2 °C). The photocycle dynamics of CaRh was investigated by sample excitation to the first inhomogeneous absorption band of the CaRhda dark-adapted state around 590 nm (long-wavelength tail), 530 nm (central region) and 470 nm (short-wavelength tail) and following the absorption spectra development during exposure and after exposure (time resolution 0.0125 s). The original protonated retinal Schiff base PRSBall-trans in CaRhda photo-converted reversibly to protonated retinal Schiff base PRSBall-trans,la1 with restructured surroundings (CaRhla1 light-adapted state, slightly blue-shifted and broadened first absorption band, recovery to CaRhda with time constant of 0.8 s) and deprotonated retinal Schiff base RSB13-cis (CaRhla2 light-adapted state, first absorption band in violet to near ultraviolet spectral region, recovery to CaRhda with time constant of 0.35 s). Long-time light exposure of light-adapted CaRhla1 around 590, 530 and 470 nm caused low-efficient irreversible degradation to photoproducts CaRhprod. Schemes of the primary photocycle dynamics of CaRhda and the secondary photocycle dynamics of CaRhla1 are developed.

Optical Control of cGMP Signaling

Zoospores of the aquatic fungi Blastocladiella emersonii are phototactic and a rhodopsin-guanylyl cyclase (RhGC) was identified as the putative phototaxis receptor. Due to the direct linkage between the rhodopsin and the cyclase domain, RhGC represents the first member of a novel class of enzyme linked rhodopsins. In this work, we characterized RhGC biophysically. We show that green light maximally activates RhGC and cyclic GMP is produced. After a short green laser flash, RhGC (D525) converts in 8 ms into a blue-shifted signaling state P380 and recovers within 100 ms. RhGC expresses well in Xenopus oocytes, yeast, CHO cells, mammalian neurons and cyclic GMP production was light dose-dependent, rapid and reproducible. Thus, RhGC is a versatile tool for optogenetic analysis of cGMP-dependent signaling processes in cell biology and the neurosciences.

Light-Activated Cyclic Nucleotide Phosphodiesterases

The second messengers 3′,5′-cyclic adenosine monophosphate (cAMP) and cyclic 3′,5′-cyclic guanosine monophosphate (cGMP) mediate various biological responses like apoptosis, smooth muscle relaxation and vision. Cyclic-nucleotide-dependent processes have been analyzed with spatiotemporal resolution through optogenetics. In particular, genetically encoded light-activated nucleotidyl cyclases have been employed to synthesize cAMP and cGMP in living cells and organisms. For example, the fungal rhodopsin-guanylyl cyclase RhGC was shown to induce light-dependent cGMP signaling in neurons. The complementary light-controlled breaking of cAMP and cGMP has been achieved through the synthetic light-activated phosphodiesterase (LAPD) only. Yet, no natural phosphodiesterase is known that catalyzes the degradation of cyclic nucleotides in a light-dependent manner. However, microbial genome and transcriptome mining lead to the emergence of natural light-activated phosphodiesterase candidates. Here we describe a natural rhodopsin-coupled phosphodiesterase (RhPD) from a flagellar protist. The final goal of this study is the isolation of a light-activated RhPD. A functional RhPD could allow optogenetic analysis of diverse cGMP/cAMP-mediated and membrane located processes in cells and vertebrate model organisms.

A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii.

Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K(+) channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K(+) selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K(+) channel and the rhodopsin-guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K(+) channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus.