Photolyase Homology Region Research Articles

Time-Resolved Ion Mobility Mass Spectrometry to Solve Conformational Changes in a Cryptochrome.

Many biological mechanisms rely on the precise control of conformational changes in proteins. Understanding such dynamic processes requires methods for determining structures and their temporal evolution. In this study, we introduce a novel approach to time-resolved ion mobility mass spectrometry. We validated the method on a simple photoreceptor model and applied it to a more complex system, the animal-like cryptochrome from Chlamydomonas reinhardtii (CraCRY), to determine the role of specific amino acids affecting the conformational dynamics as reaction to blue light activation. In our setup, using a high-power LED mounted in the source region of an ion mobility mass spectrometer, we allow a time-resolved evaluation of mass and ion mobility spectra. Cryptochromes like CraCRY are a widespread type of blue light photoreceptors and mediate various light-triggered biological functions upon excitation of their inbuilt flavin chromophore. Another hallmark of cryptochromes is their flexible carboxy-terminal extension (CTE), whose structure and function as well as the details of its interaction with the photolyase homology region are not yet fully understood and differ among different cryptochromes types. Here, we addressed the highly conserved C-terminal domain of CraCRY, to study the effects of single mutations on the structural transition of the C-terminal helix α22 and the attached CTE upon lit-state formation. We show that D321, the putative proton acceptor of the terminal proton-coupled electron transfer event from Y373, is essential for triggering the large-scale conformational changes of helix α22 and the CTE in the lit state, while D323 influences the timing.

A structural decryption of cryptochromes.

Cryptochromes (CRYs), which are signaling proteins related to DNA photolyases, play pivotal roles in sensory responses throughout biology, including growth and development, metabolic regulation, circadian rhythm entrainment and geomagnetic field sensing. This review explores the evolutionary relationships and functional diversity of cryptochromes from the perspective of their molecular structures. In general, CRY biological activities derive from their core structural architecture, which is based on a Photolyase Homology Region (PHR) and a more variable and functionally specific Cryptochrome C-terminal Extension (CCE). The α/β and α-helical domains within the PHR bind FAD, modulate redox reactive residues, accommodate antenna cofactors, recognize small molecules and provide conformationally responsive interaction surfaces for a range of partners. CCEs add structural complexity and divergence, and in doing so, influence photoreceptor reactivity and tailor function. Primary and secondary pockets within the PHR bind myriad moieties and collaborate with the CCEs to tune recognition properties and propagate chemical changes to downstream partners. For some CRYs, changes in homo and hetero-oligomerization couple to light-induced conformational changes, for others, changes in posttranslational modifications couple to cascades of protein interactions with partners and effectors. The structural exploration of cryptochromes underscores how a broad family of signaling proteins with close relationship to light-dependent enzymes achieves a wide range of activities through conservation of key structural and chemical properties upon which function-specific features are elaborated.

Long-Time Oxygen and Superoxide Localization in Arabidopsis thaliana Cryptochrome.

Cryptochromes are proteins that are highly conserved across species and in many instances bind the flavin adenine dinucleotide (FAD) cofactor within their photolyase-homology region (PHR) domain. The FAD cofactor has multiple redox states that help catalyze reactions, and absorbs photons at about 450 nm, a feature linked to the light-related functions of cryptochrome proteins. Reactive oxygen species (ROS) are produced from redox reactions involving molecular oxygen and are involved in a myriad of biological processes. Superoxide O2•- is an exemplary ROS that may be formed through electron transfer from FAD to O2, generating an electron radical pair. Although the formation of a superoxide-FAD radical pair has been speculated, it is still unclear if the required process steps could be realized in cryptochrome. Here, we present results from molecular dynamics (MD) simulations of oxygen interacting with the PHR domain of Arabidopsis thaliana cryptochrome 1 (AtCRY1). Using MD simulation trajectories, oxygen binding locations are characterized through both the O2-FAD intermolecular distance and the local protein environment. Oxygen unbinding times are characterized through replica simulations of the bound oxygen. Simulations reveal that oxygen molecules can localize at certain sites within the cryptochrome protein for tens of nanoseconds, and superoxide molecules can localize for significantly longer. This relatively long-duration molecule binding suggests the possibility of an electron-transfer reaction leading to superoxide formation. Estimates of electron-transfer rates using the Marcus theory are performed for the identified potential binding sites. Molecular oxygen binding results are compared with recent results demonstrating long-time oxygen binding within the electron-transfer flavoprotein (ETF), another FAD binding protein.

Photochemistry and Signaling Activities of Plant Cryptochromes. Review

Cryptochromes compose the widespread class of blue-light sensory receptors that in plants regulate processes of development and circadian rhythm. These photoreceptors can also function as magnetoreceptors. Cryptochrome proteins bind flavin adenine dinucleotide (FAD) as a chromophore in the photolyase homology region (PHR) domain and contain the C-terminal extension (CCE) which is joined to PHR near the FAD-binding site. The cryptochrome activation is initiated by photochemical FAD conversions involving electron/proton transfer and the formation of redox forms. In plants, cryptochrome protein with photoreduced FAD undergoes conformational changes causing disengagement of the PHR domain and CCE that is accompanied by the formation of functionally active oligomers of cryptochrome molecules. Photooligomerization is considered as a key process necessary for cryptochrome signaling activity.

Abstract 1495: Developing a model to investigate the roles of biomolecular condensates in papillary renal cell carcinoma

Abstract Cells are compartmentalized into “classical” membrane-bound organelles such as the endoplasmic reticulum and mitochondria. Cells also have membrane-less organelles (MLOs), which are formed through phase separation. Examples of MLOs include stress granules and nucleoli, where macromolecules (proteins, DNAs, RNAs) coherently and dynamically concentrate into biomolecular condensates resembling droplets in lava lamps. MLOs play central role in cellular processes - choreographed events such as transcription and RNA splicing take place in MLOs. MLOs are highly organized, and their dysregulation always led to diseases. Papillary renal cell carcinoma (PRCC) is the second most common RCC in America. PRCC is an aggressive cancer, yet the uncharacterized molecular etiology results in the lack of effective treatment for patients. Our lab and others have observed dysregulated oncoproteins MLOs in PRCC. However, the molecular properties and functions of these aberrant MLOs have not been characterized. The photolyase homology region of Arabidopsis thaliana Cry2 (Cry2WT) is a light-sensitive protein that self-associate with blue light exposure. We hypothesize that fusing dysregulated MLOs oncoproteins with Cry2WT will allow us to tune and study the oncoproteins condensates. We aim to develop an optogenetic model to study how oncoproteins condensates function in PRCC cells. Dysregulated oncoproteins were fused to mCherry-Cry2WT, introduced into cell line models, light-activated and imaged using a ZEISS LSM 900 microscope. We found that blue-light indeed induced proteins clustering when oncoproteins were fused to mCherry-Cry2WT but not with mCherry-Cry2WT alone. This model will allow us to tune the formation of oncoproteins condensates. Future work will involve studying how optogenetically tuned oncoproteins condensates function in PRCC patient-derived cell lines. Citation Format: Choon Leng So, Jane Lee, Binglin Huang, Danfeng Cai. Developing a model to investigate the roles of biomolecular condensates in papillary renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1495.

Direct experimental observation of blue-light-induced conformational change and intermolecular interactions of cryptochrome

Cryptochromes are blue light receptors that mediate circadian rhythm and magnetic sensing in various organisms. A typical cryptochrome consists of a conserved photolyase homology region domain and a varying carboxyl-terminal extension across species. The structure of the flexible carboxyl-terminal extension and how carboxyl-terminal extension participates in cryptochrome’s signaling function remain mostly unknown. In this study, we uncover the potential missing link between carboxyl-terminal extension conformational changes and downstream signaling functions. Specifically, we discover that the blue-light induced opening of carboxyl-terminal extension in C. reinhardtii animal-like cryptochrome can structurally facilitate its interaction with Rhythm Of Chloroplast 15, a circadian-clock-related protein. Our finding is made possible by two technical advances. Using single-molecule Förster resonance energy transfer technique, we directly observe the displacement of carboxyl-terminal extension by about 15 Å upon blue light excitation. Combining structure prediction and solution X-ray scattering methods, we propose plausible structures of full-length cryptochrome under dark and lit conditions. The structures provide molecular basis for light active conformational changes of cryptochrome and downstream regulatory functions.

Assessing the Phase Separation Propensity of Proteins in Living Cells Through Optodroplet Formation.

Phase separation is emerging as a key mechanism to describe the formation of membraneless organelles in the cell. It depends on the multivalent (self-) interaction properties of the macromolecules involved and can be observed in aqueous solutions under controlled conditions in vitro with purified components. However, to experimentally demonstrate that this process indeed occurs in the complex environment of living cells remains difficult. Here, we describe an assay based on light-induced association of proteins into complexes termed optodroplets that are in the hundred nm to μm size range. The formation and dissociation of these optodroplets can be followed over time in living cells by fluorescence microscopy to evaluate the propensity of proteins to demix and to form phase-separated subcompartments. The optodroplet assay is based on the fusion of a protein of interest with the photolyase homology region (PHR) protein domain from Arabidopsis thaliana, which can undergo reversible homo-oligomerization upon illumination with blue light. Using this approach, candidate proteins and their interaction-deficient or interaction-enhanced variants can be compared to each other or to reference proteins with known phase separation features. By quantifying the resulting microscopy images, the propensity of a given protein construct to assemble into a phase-separated subcompartment can be assessed.

Plant Cryptochromes Illuminated: A Spectroscopic Perspective on the Mechanism.

Plant cryptochromes are central blue light receptors for the control of land plant and algal development including the circadian clock and the cell cycle. Cryptochromes share a photolyase homology region with about 500 amino acids and bind the chromophore flavin adenine dinucleotide. Characteristic for plant cryptochromes is a conserved aspartic acid close to flavin and an exceptionally long C-terminal extension. The mechanism of activation by excitation and reduction of the chromophore flavin adenine dinucleotide has been controversially discussed for many years. Various spectroscopic techniques have contributed to our understanding of plant cryptochromes by providing high time resolution, ambient conditions and even in-cell approaches. As a result, unifying and differing aspects of photoreaction and signal propagation have been revealed in comparison to members from other cryptochrome subfamilies. Here, we review the insight from spectroscopy on the flavin photoreaction in plant cryptochromes and present the current models on the signal propagation from flavin reduction to dissociation of the C-terminal extension.

C-Terminal Extension of a Plant Cryptochrome Dissociates from the β-Sheet of the Flavin-Binding Domain.

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting β-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the β-sheet signal, implying an association of the CCT close to the only β-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that β-sheet reorganization causes the CCT release and restructuring.

Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock.

CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective and spatiotemporally controllable tools will facilitate the understanding of shared and distinct functions of CRY1 and CRY2. Here, we developed CRY1-selective compounds that enable light-dependent manipulation of the circadian clock. From phenotypic chemical screening in human cells, we identified benzophenone derivatives that lengthened the circadian period. These compounds selectively interacted with the CRY1 photolyase homology region, resulting in activation of CRY1 but not CRY2. The benzophenone moiety rearranged a CRY1 region called the “lid loop” located outside of the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis–trans isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light. This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.

The human CRY1 tail controls circadian timing by regulating its association with CLOCK:BMAL1

Circadian rhythms are generated by interlocked transcription-translation feedback loops that establish cell-autonomous biological timing of ∼24 h. Mutations in core clock genes that alter their stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR-tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR-tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity.

Regulatory Impact of the C-Terminal Tail on Charge Transfer Pathways in Drosophila Cryptochrome.

Interconnected transcriptional and translational feedback loops are at the core of the molecular mechanism of the circadian clock. Such feedback loops are synchronized to external light entrainment by the blue light photoreceptor cryptochrome (CRY) that undergoes conformational changes upon light absorption by an unknown photoexcitation mechanism. Light-induced charge transfer (CT) reactions in Drosophila CRY (dCRY) are investigated by state-of-the-art simulations that reveal a complex, multi-redox site nature of CT dynamics on the microscopic level. The simulations consider redox-active chromophores of the tryptophan triad (Trp triad) and further account for pathways mediated by W314 and W422 residues proximate to the C-terminal tail (CTT), thus avoiding a pre-bias to specific W-mediated CT pathways. The conducted dissipative quantum dynamics simulations employ microscopically derived model Hamiltonians and display complex and ultrafast CT dynamics on the picosecond timescale, subtly balanced by the electrostatic environment of dCRY. In silicio point mutations provide a microscopic basis for rationalizing particular CT directionality and demonstrate the degree of electrostatic control realized by a discrete set of charged amino acid residues. The predicted participation of CT states in proximity to the CTT relates the directionality of CT reactions to the spatial vicinity of a linear interaction motif. The results stress the importance of CTT directional charge transfer in addition to charge transfer via the Trp triad and call for the use of full-length CRY models including the interactions of photolyase homology region (PHR) and CTT domains.

CL6mN: Rationally Designed Optogenetic Photoswitches with Tunable Dissociation Dynamics.

The field of optogenetics uses genetically encoded photoswitches to modulate biological phenomena with high spatiotemporal resolution. We report a set of rationally designed optogenetic photoswitches that use the photolyase homology region of A.thaliana cryptochrome 2 (Cry2PHR) as a building block and exhibit highly efficient and tunable clustering in a blue-light dependent manner. CL6mN (Cry2-mCherry-LRP6c with N mutated PPPAP motifs) proteins were designed by mutating and/or truncating five crucial PPP(S/T)P motifs near the C-terminus of the optogenetic Wnt activator Cry2-mCherry-LRP6c, thus eliminating its Wnt activity. Light-induced CL6mN clusters have significantly greater dissociation half-lives than clusters of wild-type Cry2PHR. Moreover, the dissociation half-lives can be tuned by varying the number of PPPAP motifs, with the half-life increasing as much as 6-fold for a variant with five motifs (CL6m5) relative to Cry2PHR. Finally, we demonstrate the compatibility of CL6mN with previously reported Cry2-based photoswitches by optogenetically activating RhoA in mammalian cells.

Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

A Tail of Night Owls: The Mammalian Cry1 C-Terminal Tail Controls Circadian Timing by Regulating its Association to CLOCK:BMAL1

Circadian rhythms are generated by a transcription-translation feedback loop that establishes cell-autonomous biological timing of ∼24-hours. A prevalent human variation in the core clock gene cryptochrome 1, Cry1Δ11, lengthens circadian period to cause Delayed Sleep Phase Disorder (DSPD). CRY1 has a 55 kDa photolyase homology region (PHR) followed by a ∼100 residue tail that is intrinsically disordered; the Δ11 variant lacks a short segment encoded by Exon 11 within its tail. Using fluorescence polarization binding assays and carbon detected NMR spectroscopy, we show here that the disordered tail of CRY1 interacts directly with its PHR, Furthermore we use fluorescence polarization and biolayer interferometry to show that Exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and CLOCK, a subunit of the primary circadian transcription factor. Competition between PER2 and the tail for the CRY1 PHR suggests a regulatory role for the tail in the early morning, when CRY1 binds to CLOCK:BMAL1 on DNA independently of PER2. Discovery of this autoregulatory role for mammalian CRY1 highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity.

Structural changes within the bifunctional cryptochrome/photolyase CraCRY upon blue light excitation

Cryptochromes (CRYs) are an ubiquitously occurring class of photoreceptors, which are important for regulating the circadian rhythm of animals via a time-delayed transcription-translation feedback loop (TTFL). Due to their protein architecture and common FAD chromophore, they belong to the same superfamily as photolyases (PHLs), an enzyme class that repairs UV-induced DNA lesions upon blue light absorption. Apart from their different functions the only prominent structural difference between CRY and PHL is the highly variable C-terminal extension (CTE) of the former. The nature of the CTE is still unclear and highly speculated. In this study, we show by hydrogen/deuterium exchange and subsequent mass-spectrometric analysis that the CTE of the animal-like cryptochrome from the green algae Chlamydomonas reinhardtii (CraCRY) binds to the surface of the photolyase homology region, which flanks the DNA binding site. We also compared the fully oxidized and fully reduced states of the flavoprotein and designed a tool, so called light chamber, for automated HDX-MS measurements of photoreceptors in defined photostates. We could observe some striking differences between the two photostates and propose a model for light-dependent switching of this bifunctional cryptochrome.

Light sensitivity of the photoreceptor cryptochrome of the Drosophila circadian clock and its interaction with other clock components

Flavoprotein cryptochromes (CRYs) are blue light sensors that regulate the circadian clock in plants and animals. They consist of a highly conserved photolyase homology region (PHR) and a C‐terminal tail extension (CTE) of various sizes. In Drosophila CRY, photoreduction of the PHR bound cofactor FAD triggers conformational changes within CRY, which include undocking of C‐terminal Tail (CTT) helix. The mechanism of FAD photoreduction remains a key question in understanding CRY activation.Four tryptophan residues (W420, W397, W342, W394) mediate electron transfer for flavin photoreduction. We have substituted these residues to both redox‐inert phenylalanine residues (F) and redox‐active tyrosine (Y) residues to tune the light sensitivity of CRY. Light‐sensitivities of the variants correlate well with the extent of conformational change at the CTT. Interestingly, the surface tryptophan W394 is the most indispensable residue because it gates electron transfer from exogenous reductant to the photoreduction chain.In flies, CRY interacts with other signaling proteins of the circadian clock in a light depend‐manner to entrain the 24 hour‐cycle to environmental light. For example, in light CRY binds the protein timeless (TIM) and recruits the E3 ligase jetlag to facilitate TIM degradation. However, the molecular interactions between CRY and TIM are not well understood due to the low stability of this protein complex. We have utilized a chemical cross‐linking strategy to map the interfacing domain in this assembly and stabilize it for future structure study by structural methodsSupport or Funding InformationThis work was supported by grants from the NIH: R01GM054339 (to M.W.Y.) and R35GM122535 (to B.R.C.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impacts of Cys392, Asp393, and ATP on the FAD Binding, Photoreduction, and the Stability of the Radical State of Chlamydomonas reinhardtii Cryptochrome.

Plant cryptochromes (CRYs) are blue-light receptors that regulate light-dependent growth, development, and circadian rhythms. A flavin adenine dinucleotide (FAD) cofactor is bound to the photolyase homology region (PHR) of plant CRYs and can be photoreduced to a neutral radical state under blue light. This photoreaction can trigger subsequent signal transduction. Plant CRYs can also bind an ATP molecule adjacent to FAD in a pocket of the PHR. Chlamydomonas reinhardtii contains a single plant CRY, named Chlamydomonas photolyase homologue 1 (CPH1). In CPH1, Cys392 and Asp393 are located near the FAD cofactor. Here we have shown that replacing Cys392 with Ser has little effect on the properties of CPH1. The C392N mutant, however, showed a faster photoreduction rate than wild-type CPH1, together with a significantly lower oxidation rate of the neutral radical state. Substituting an Asn residue for Asp393 in CPH1 improved the binding affinity for FAD as well as the stability of the neutral radical, but photoreduction in the case of this mutant was severely inhibited. In the presence of ATP, CPH1 and its mutants exhibited significantly higher binding affinity for FAD and slower oxidation of the neutral radical. These results reveal that the residues at site 392 and the presence of ATP can tune the stability of the neutral radical, that the Asp residue at site 393 is crucial for photoreduction, and that the photoreduction rate is not determined merely by the stability of the neutral radical in CPH1.

Shedding Light on Night Owl Behavior: How the Disordered C-Terminal Tail of CRY1 Modulates Circadian Timekeeping

Circadian rhythms are driven by a negative feedback loop in which the heterodimer CLOCK:BMAL1 promotes transcription of clock-controlled genes, including its own repressors Period (PER) and Cryptochrome (CRY). The deregulation of circadian rhythms can lead to delayed sleep phase disorder (DSPD), where the afflicted “night owls” are only able to fall asleep hours after midnight and have difficulty waking up early in the morning. CRY proteins contain a photolyase homology region (PHR) and an intrinsically disordered C-terminal tail; previous studies identified an essential role for the CRY1 PHR in generating circadian rhythms, while deletion of the tail was shown to alter the length of the circadian period. Our lab recently identified how the CRY1 PHR binds to both CLOCK and BMAL1 to induce repression and close the negative feedback loop. To date, little is known about how the CRY1 PHR and tail might interact to control this process. One prevalent mutation in DSPD patients leads to deletion of CRY1 Exon 11, a region that encodes for 25 amino acids in the tail, resulting in enhanced co-immunoprecipitation by CLOCK:BMAL1 and a longer circadian period. We hypothesized that the CRY1 tail might bind directly to the PHR to regulate interactions with CLOCK or BMAL1. Here we show that the C-terminal tail and CLOCK compete for binding to the CRY1 PHR, and that the peptide encoded by Exon 11 is sufficient for this interaction. Therefore, our data demonstrate how the tail can directly regulate CRY1 interactions with CLOCK:BMAL1 to provide initial insight into the molecular basis of DSPD.

Time-Resolved Infrared Spectroscopy on Plant Cryptochrome-Relevance of Proton Transfer and ATP Binding for Signaling.

Plant cryptochromes are light receptors in land plants and algae with very diverse functions such as circadian timing and lifecycle progression. The receptor consists of a photolyase homology region (PHR) binding the flavin chromophore and a C-terminal extension (CCT) responsible for signaling. The reputed signaling state, the flavin neutral radical, is formed by a femtosecond electron transfer and microsecond proton transfer to the excited, oxidized flavin. Subsequently, a 500 μs loss of β-sheet structure ∼25 Å away from flavin was resolved and suggested to be part of the signal conduction to the CCT. Here, we performed time-resolved, step-scan Fourier transform IR spectroscopy on the PHR of the plant cryptochrome pCRY (formerly CPH1) from Chlamydomonas reinhardtii. In a mutant lacking the proton donor aspartic acid 396 only the flavin anion radical is formed, but we observed the loss of β-sheet structure with a time constant of 1.3 ms, similar to the 500 μs of the wild type. This finding implies that the anion radical may be considered signaling-competent. In the steady state, a variation of external pH up to 8.3 did not have any effect on the difference spectra including the protonated state of Asp396. However, we detected the prominent loss of β-sheet structure by illumination only in the presence of adenosine triphosphate (ATP). We conclude that the bound ATP stabilizes these light-induced changes in secondary structure to ensure a physiological lifetime compatible with signaling by plant cryptochrome.