Revisiting and Redesigning Light-Activated Cyclic-Mononucleotide Phosphodiesterases

Stabel, Robert; Stüven, Birthe; Hansen, Jan N.; Körschen, Heinz G.; Wachten, Dagmar; Möglich, Andreas

doi:10.1016/j.jmb.2019.07.011

Cited by 23 publications

(30 citation statements)

References 68 publications

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“…Where present in the structure, the effectors are mostly connected to the PCM via a continuous parallel coiled coil, thus possibly restricting the quaternary structures the PCM can adopt in the Pr and Pfr states. In particular, it remains to be seen whether the large‐scale splaying apart of the PHY domains observed for the isolated Dr BPhy PCM manifests to the same extent within the context of full‐length native and engineered BPhy receptors . In fact, recent structures of a BPhy‐GGDEF enzyme indicate that the conformational transitions within the context of a full‐length receptor are of much smaller amplitude .…”

Section: Photosensory Inputmentioning

confidence: 99%

“…In particular, it remains to be seen whether the large-scale splaying apart of the PHY domains observed for the isolated DrBPhy PCM manifests to the same extent within the context of fulllength native and engineered BPhy receptors. 105,[111][112][113][114][115] In fact, recent structures of a BPhy-GGDEF enzyme indicate that the conformational transitions within the context of a fulllength receptor are of much smaller amplitude. 107 Regardless, the long continuous α helices are conducive to downstream relay of the conformational signal, see below.…”

Section: Bacteriophytochrome Signalingmentioning

confidence: 99%

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Signal transduction in photoreceptor histidine kinases

Möglich

2019

Protein Science

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Two‐component systems (TCS) constitute the predominant means by which prokaryotes read out and adapt to their environment. Canonical TCSs comprise a sensor histidine kinase (SHK), usually a transmembrane receptor, and a response regulator (RR). In signal‐dependent manner, the SHK autophosphorylates and in turn transfers the phosphoryl group to the RR which then elicits downstream responses, often in form of altered gene expression. SHKs also catalyze the hydrolysis of the phospho‐RR, hence, tightly adjusting the overall degree of RR phosphorylation. Photoreceptor histidine kinases are a subset of mostly soluble, cytosolic SHKs that sense light in the near‐ultraviolet to near‐infrared spectral range. Owing to their experimental tractability, photoreceptor histidine kinases serve as paradigms and provide unusually detailed molecular insight into signal detection, decoding, and regulation of SHK activity. The synthesis of recent results on receptors with light‐oxygen‐voltage, bacteriophytochrome and microbial rhodopsin sensor units identifies recurring, joint signaling strategies. Light signals are initially absorbed by the sensor module and converted into subtle rearrangements of α helices, mostly through pivoting and rotation. These conformational transitions propagate through parallel coiled‐coil linkers to the effector unit as changes in left‐handed superhelical winding. Within the effector, subtle conformations are triggered that modulate the solvent accessibility of residues engaged in the kinase and phosphatase activities. Taken together, a consistent view of the entire trajectory from signal detection to regulation of output emerges. The underlying allosteric mechanisms could widely apply to TCS signaling in general.

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Section: Photosensory Inputmentioning

confidence: 99%

Section: Bacteriophytochrome Signalingmentioning

confidence: 99%

Signal transduction in photoreceptor histidine kinases

Möglich

2019

Protein Science

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“…Ca 2+ imaging in 96-well plates in a fluorescence plate reader was performed as previously described (Jansen et al, 2015, Stabel et al, 2019. A detailed protocol and all changes are described in the SI Appendix.…”

Section: Ca 2+ Imagingmentioning

confidence: 99%

“…The photo-activated adenylate cyclase bPAC (Stierl, Stumpf et al, 2011) has been employed to increase flagellar cAMP levels by blue light (Jansen et al, 2015), and the FRET-based cAMP biosensor mlCNBD-FRET has been used to monitor cAMP dynamics in sperm flagella (Mukherjee et al, 2016). This cAMP tool kit can be complemented with the red light-activated phosphodiesterase LAPD that allows to decrease cAMP levels in a light-dependent manner (Gasser, Taiber et al, 2014, Stabel, Stüven et al, 2019. In cells other than sperm, the challenge is to specifically target these tools to the primary cilium to investigate cAMP signaling in the cilium, but not in the cell body.…”

Section: Introductionmentioning

confidence: 99%

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Hansen

Kaiser

Klausen

et al. 2020

Preprint

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Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. Primary cilia constitute a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling and function has been challenging due to the lack of tools to manipulate and analyze ciliary signaling in living cells. Here, we describe a nanobodybased targeting approach for optogenetic tools that allows to specifically analyze ciliary signaling and function, and that is applicable in vitro and in vivo. We overcome the loss of protein function observed after direct fusion to a ciliary targeting sequence, and functionally localize the photo-activated adenylate cyclase bPAC, the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we unravel the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

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“…Even prior to the present elucidation of the enzymatic activity of DrBphP, its PSM has provided a versatile building block for novel light-controllable enzymes to be used in optogenetics. Pertinent enzymes have for instance been constructed through fusion of the DrBphP PSM with a cyclicmononucleotide phosphodiesterase 56,57 , a guanylate/adenylate cyclase 58,59 , and a tyrosine kinase 60 . Our study introduces a red light-regulated HK chimera and phosphatase as a potential addition to the optogenetic toolkit.…”

Section: The Bacteriophytochrome From D Radiodurans Is a Light-activmentioning

confidence: 99%

Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

Multamäki

Nanekar

Morozov

et al. 2020

Preprint

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Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this longstanding conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (AgP1). Whereas AgP1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While AgP1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes inform the use of light-controllable histidine kinases and phosphatases for optogenetics. radiodurans (DrBphP), light induces extensive structural changes in the photosensory module that are relayed to the output module 29 .In terms of enzymatic activity, the dark-adapted Pr state exhibited higher kinase activity than the Pfr state in cyanobacterial phytochrome Cph1, 27,30,31 , similar to other bacteriophytochromes 32 . In particular, the bacteriophytochrome from Agrobacterium fabrum (AgP1) displays histidine kinase activity in its resting Pr state 28,33 ; in the Pfr state, the autophosphorylation and phosphotransfer reactions are down-regulated by 2-fold and 10-fold, respectively 28 . This kinase activity of the AgP1 has been shown to control bacterial conjugation 34 . By contrast, no kinase activity has been demonstrated for DrBphP, notwithstanding close sequence homology and the elaborate structural changes this receptor undergoes under light 22,29 . Despite the eminent role of DrBphP as a paradigm for photoreception, the enzymatic activity and the physiological role of this model phytochrome have hence remained enigmatic.Here, we unravel this long-standing puzzle by studying the enzymatic activity and interactions of DrBphP and AgP1, as two canonical bacteriophytochromes with HK effector domains. By pursuing an integrated biochemical and structural strategy, we show that despite close homology, AgP1 acts as a histidine kinase whereas DrBphP functions as a light-activated phosphatase. Our biochemical and structural data pinpoint two key residues proximal to the catalytic histidine that affect the balance between the kinase and phosphatase activities. Together, the two phytochromes provide soluble, lightcontrollable systems with opposite activities for the study and application of two-component signaling.

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Revisiting and Redesigning Light-Activated Cyclic-Mononucleotide Phosphodiesterases

Cited by 23 publications

References 68 publications

Signal transduction in photoreceptor histidine kinases

Signal transduction in photoreceptor histidine kinases

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

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