Resonance Raman Investigation of the Chromophore Structure of Heliorhodopsins

Otomo, Akira; Mizuno, Misao; Singh, Manish Pratap; Shihoya, Wataru; Inoue, Kazuhide; Nureki, Osamu; Béjà, Oded; Kandori, Hideki; Mizutani, Yasuhisa

doi:10.1021/acs.jpclett.8b02741

“…This could be observed as well in the case of HeR-48C12 and the absence of ion transport activity for both BcHeR and HeR-48C12 may indicate that the heliorhodopsin family has other functions such as a light-sensor or the light-induced transport of molecules such as glycopeptides or amphiphilic molecules as proposed in previous studies. 18,27,34 We next characterized the absorption spectra and retinal configuration of purified BcHeR in dark-and in light-illuminated conditions. Many microbial rhodopsins and even HeR-48C12 present not only a light-adapted state 18 but also a dark-adapted state.…”

Section: Resultsmentioning

confidence: 99%

Photochemical Characterization of a New Heliorhodopsin from the Gram-Negative Eubacterium Bellilinea caldifistulae (BcHeR) and Comparison with Heliorhodopsin-48C12

Shibukawa

¹

,

Kojima

²

,

Nakajima

³

et al. 2019

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Many microorganisms express rhodopsins, pigmented membrane proteins capable of absorbing sunlight and harnessing that energy for important biological functions such as ATP synthesis and phototaxis. Microbial rhodopsins that have been discovered to date are categorized as type-1 rhodopsins. Interestingly, researchers have very recently unveiled a new microbial rhodopsin family named heliorhodopsins that are phylogenetically distant from type-1 rhodopsins. Among them, only heliorhodopsin-48C12 (HeR-48C12) from a gram-positive eubacterium has been photochemically characterized [Pushkarev et al., 2018, Nature, 558, 595-599]. In this study, we photochemically characterize a purple-colored heliorhodopsin from gram-negative eubacterium Bellilinea caldifistulae (BcHeR) as a second example and we identified which properties are or are not conserved between BcHeR and HeR-48C12. A series of photochemical measurements revealed several conserved properties between them, including a visible absorption spectrum with a maximum at around 550 nm, the lack of ion-transport activity, and the existence of a second-order O-like intermediate during the photocycle that may activate an unidentified biological function. In contrast, as a property that is not conserved, although HeR-48C12 shows the light-adaptation state of retinal, BcHeR showed the same retinal configuration both in darkand in light-adapted conditions. These comparisons of photochemical properties between BcHeR and HeR-48C12 are an important first step toward understanding the nature and functional role of heliorhodopsins.

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“…Rhodopsins commonly have seven TMs and the retinal chromophore. Recently, a distinct and abundant group of microbial rhodopsins, HeRs, has been discovered [37][38][39] . Although HeRs share low sequence identity with the other rhodopsins and adopt the inverted membrane topology, the crystal structures revealed that HeR comprises seven TMs with the retinal chromophore, as in the other rhodopsins [40][41][42] .…”

Section: Discussionmentioning

confidence: 99%

Structural insights into the mechanism of rhodopsin phosphodiesterase

Ikuta

¹

,

Shihoya

²

,

Sugiura

³

et al. 2020

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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.

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“…Rhodopsins commonly have 7 TMs and the retinal chromophore. Recently, a distinct and abundant group of microbial rhodopsins, the heliorhodopsins (HeRs) has been discovered [36][37][38] . Although HeRs share low sequence identity with the other rhodopsins and adopt the inverted membrane topology, the crystal structures revealed that HeR comprises 7…”

Section: Discussionmentioning

confidence: 99%

“…This study deepens our knowledge about the structures and functions of enzyme rhodopsins, and will facilitate the design of a dark activity-reduced Rh-PDE for optogenetic applications.Rhodopsins commonly have 7 TMs and the retinal chromophore. Recently, a distinct and abundant group of microbial rhodopsins, the heliorhodopsins (HeRs) has been discovered [36][37][38] . Although HeRs share low sequence identity with the other rhodopsins and adopt the inverted membrane topology, the crystal structures revealed that HeR comprises 7TMs with the retinal chromophore, as in the other rhodopsins [39][40][41] .…”

mentioning

confidence: 99%

Structural insights into the mechanism of rhodopsin phosphodiesterase

Ikuta

¹

,

Shihoya

²

,

Sugiura

³

et al. 2020

Preprint

Self Cite

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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 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. -3 - Main text IntroductionMicrobial rhodopsins are photoreceptive membrane proteins with an all-trans retinylidene chromophore 1,2 (all-trans retinal). All microbial rhodopsins share a seven transmembrane topology, in which all-trans retinal chromophore is bound to a conserved lysine residue via Schiff base in the transmembrane helix (TM) 7. Most microbial rhodopsins function as light-driven ion pumps that actively transport various ions 1 (H + , Na + , Cl -, etc.). Some microbial rhodopsins function as light-gated ion channels (channelrhodopsins 3,4 ), which are used as optogenetic tools in neuroscience 5,6 . Enzyme rhodopsins are a group of newly discovered microbial rhodopsins 7 , found in eukaryotes such as fungi and green algae, and choanoflagellates. Enzyme rhodopsins comprise the N-terminal rhodopsin domain and C-terminal enzyme domain, connected by a linker, and function as light-activated enzymes. Two types of enzyme rhodopsins were initially discovered, the histidine kinase rhodopsins 8 (HKRs) and rhodopsin guanylyl cyclase 9 (Rh-GC). While HKRs function as ATP-dependent light-inhibited guanylyl cyclase 10 , Rh-GC from Blastocladiella emersonii (BeGC1) induces a rapid light-triggered cGMP increase in heterologous cells (~5,000-fold) and could be utilized as an optogenetic tool to rapidly manipulate cGMP levels in cells and animals 11,12 .In 2017, rhodopsin phosphodiesterase 13 (Rh-PDE), a novel type of enzyme rhodopsin, was discovered. Rh-PDE derived from Salpingoeca rosetta (SrRh-PDE) was first identified and exhibits light-dependent hydrolytic activity for both of cAMP and cGMP, whereas it lacks pump or channel activity. Moreover, four Rh-PDE homologs were recently discovered in Choanoeca flexa 14 . Thes...

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Resonance Raman Investigation of the Chromophore Structure of Heliorhodopsins

Cited by 37 publications

References 41 publications

Photochemical Characterization of a New Heliorhodopsin from the Gram-Negative Eubacterium Bellilinea caldifistulae (BcHeR) and Comparison with Heliorhodopsin-48C12

Photochemical Characterization of a New Heliorhodopsin from the Gram-Negative Eubacterium Bellilinea caldifistulae (BcHeR) and Comparison with Heliorhodopsin-48C12

Structural insights into the mechanism of rhodopsin phosphodiesterase

Structural insights into the mechanism of rhodopsin phosphodiesterase

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