Photoactivation Mechanism of a Bacterial Light-Regulated Adenylyl Cyclase

Lindner, Robert; Hartmann, Elisabeth; Tarnawski, Mirosław; Winkler, Andreas; Frey, Daniel; Reinstein, Jochen; Meinhart, Anton; Schlichting, Ilme

doi:10.1016/j.jmb.2017.03.020

Cited by 51 publications

(121 citation statements)

References 53 publications

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“…Based on this result, we conclude that photoactivation of cPAC does not lead to a major change in overall protein conformation. This also implies that small lightinduced structural changes are responsible for the regulation of activity of cPAC, a result similar to that reported for BLUF containing PACs (11,37,38). Our HPLC-SEC data also suggests that homodimerization is required for efficient catalytic turnover.…”

Section: Cyanobacterial Photoactivatable Adenylyl Cyclasessupporting

confidence: 87%

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells

Blain-Hartung

Rockwell

Moreno

et al. 2018

Journal of Biological Chemistry

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Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in nonphotosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium sp. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibits a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for the design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells, tissues, and whole organisms with light across the visible spectrum and into the near IR.

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Section: Cyanobacterial Photoactivatable Adenylyl Cyclasessupporting

confidence: 87%

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells

Blain-Hartung

Rockwell

Moreno

et al. 2018

Journal of Biological Chemistry

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“…The amino acids tyrosine (Y), asparagine (N), glutamine (Q), and tryptophan (W) or methionine (M), which are crucial for the photodynamics and photocycle of the BLUF domain, are highly conserved ( Figure 2). Our analysis further confirms that tyrosine, glutamine, and tryptophan (or methionine) are critical for the substrate specificity of the BLUF sequences, as has been reported earlier [6,11,33,57]. The photo-activation of the BLUF domains actually involves conserved glutamine and tyrosine residues, where glutamine contributes to hydrogen bond formation in a dark state [11][12][13][14].…”

Section: Bluf Sequences Modular Domains and Phylogenetic Analysissupporting

confidence: 89%

“…The function of these short auxillary helical stretches, located in the association of several BLUF and LOV photoreceptors, is not known precisely, but it seems likely that they mediate the signal progression between their photosensors and the effector domain, as has been predicted in earlier reports [62]. We modeled the binding modes of the flavin chromophore based on the observations in the known BLUF domain structures [33,57]. The isoalloxazine ring of flavin can be readily accommodated in the pocket in the models sandwiched between two α helices, suggesting that the active site is appropriately formed in BLUF models (Figures 4 and 5).…”

Section: Modular Diversity Of Bluf Domainsmentioning

confidence: 95%

“…The BLUF domains reveal conserved βαββαββ fold conformations in which two α-helices surround a five-stranded antiparallel β-sheet platform (Figure 4). We modeled the binding modes of the flavin chromophore based on the observations in the known BLUF domain structures [33,57]. The isoalloxazine ring of flavin can be readily accommodated in the pocket in the models sandwiched between two α helices, suggesting that the active site is appropriately formed in BLUF models (Figures 4 and 5).…”

Section: Modular Diversity Of Bluf Domainsmentioning

confidence: 99%

“…Furthermore, we have performed a alignment of sequences representing the CHD domain from selected proteins against the progression of the well-characterized template CHD domain of bPAC ( Figure S2). The multiple sequence alignment revealed that active site residues involved in the nucleotide binding (i.e., Asn257-His266, Lys263-Met264 (forming the β4AC-β5AC tongue), Gly259-Asn178 (forming the α2AC helix) and Lys263-Thr196, Asp265-Phe198, and His266-Lys197 (forming the β2AC-β3AC hairpin) [33]) are highly conserved among all three selected BLUF-CHD domain-containing proteins ( Figure S2). In PACs, under dark conditions, the orientation and arrangement of an individual amino acid in the active site (i.e., Thr267 and Lys197 (bound to Phe198)) render the conformation of AC inactive.…”

Section: Cyclase Homology Domain (Chd)mentioning

confidence: 99%

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Modular Diversity of the BLUF Proteins and Their Potential for the Development of Diverse Optogenetic Tools

et al. 2019

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Organisms can respond to varying light conditions using a wide range of sensory photoreceptors. These photoreceptors can be standalone proteins or represent a module in multidomain proteins, where one or more modules sense light as an input signal which is converted into an output response via structural rearrangements in these receptors. The output signals are utilized downstream by effector proteins or multiprotein clusters to modulate their activity, which could further affect specific interactions, gene regulation or enzymatic catalysis. The blue-light using flavin (BLUF) photosensory module is an autonomous unit that is naturally distributed among functionally distinct proteins. In this study, we identified 34 BLUF photoreceptors of prokaryotic and eukaryotic origin from available bioinformatics sequence databases. Interestingly, our analysis shows diverse BLUF-effector arrangements with a functional association that was previously unknown or thought to be rare among the BLUF class of sensory proteins, such as endonucleases, tet repressor family (tetR), regulators of G-protein signaling, GAL4 transcription family and several other previously unidentified effectors, such as RhoGEF, Phosphatidyl-Ethanolamine Binding protein (PBP), ankyrin and leucine-rich repeats. Interaction studies and the indexing of BLUF domains further show the diversity of BLUF-effector combinations. These diverse modular architectures highlight how the organism’s behaviour, cellular processes, and distinct cellular outputs are regulated by integrating BLUF sensing modules in combination with a plethora of diverse signatures. Our analysis highlights the modular diversity of BLUF containing proteins and opens the possibility of creating a rational design of novel functional chimeras using a BLUF architecture with relevant cellular effectors. Thus, the BLUF domain could be a potential candidate for the development of powerful novel optogenetic tools for its application in modulating diverse cell signaling.

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An Integrated Optogenetic and Bioelectronic Platform for Regulating Cardiomyocyte Function

Bolonduro,

Chen,

Fucetola

et al. 2024

Advanced Science

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Bioelectronic medicine is emerging as a powerful approach for restoring lost endogenous functions and addressing life‐altering maladies such as cardiac disorders. Systems that incorporate both modulation of cellular function and recording capabilities can enhance the utility of these approaches and their customization to the needs of each patient. Here is report an integrated optogenetic and bioelectronic platform for stable and long‐term stimulation and monitoring of cardiomyocyte function in vitro. Optical inputs are achieved through the expression of a photoactivatable adenylyl cyclase, that when irradiated with blue light causes a dose‐dependent and time‐limited increase in the secondary messenger cyclic adenosine monophosphate with subsequent rise in autonomous cardiomyocyte beating rate. Bioelectronic readouts are obtained through a multi‐electrode array that measures real‐time electrophysiological responses at 32 spatially‐distinct locations. Irradiation at 27 µW mm−2 results in a 14% elevation of the beating rate within 20–25 min, which remains stable for at least 2 h. The beating rate can be cycled through “on” and “off” light states, and its magnitude is a monotonic function of irradiation intensity. The integrated platform can be extended to stretchable and flexible substrates, and can open new avenues in bioelectronic medicine, including closed‐loop systems for cardiac regulation and intervention, for example, in the context of arrythmias.

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Photoactivation Mechanism of a Bacterial Light-Regulated Adenylyl Cyclase

Cited by 51 publications

References 53 publications

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells

Modular Diversity of the BLUF Proteins and Their Potential for the Development of Diverse Optogenetic Tools

An Integrated Optogenetic and Bioelectronic Platform for Regulating Cardiomyocyte Function

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