Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome

Lin, Changfan; Schneps, Connor M; Chandrasekaran, Siddarth; Ganguly, Abir; Crane, Brian R.

doi:10.1016/j.str.2022.03.010

Cited by 15 publications

(35 citation statements)

References 54 publications

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“…3a). CRY purifies as a monomer (Berndt et al, 2007;Zoltowski et al, 2011;Levy et al, 2013) and there is no evidence that it dimerizes in cells (Lin et al, 2022). An F o À F c omit map reveals strong positive density for FAD within the -helical domain (Fig.…”

Section: The Overall Structure Is Similar At Cryogenic and Room Tempe...mentioning

confidence: 99%

“…CRY serves as the principal photoreceptor that entrains the circadian clock to light (Emery et al, 1998;Stanewsky et al, 1998;Chaves et al, 2011). In response to blue light, the oxidized FAD cofactor is photoreduced to the negatively charged anionic semiquinone (Vaidya et al, 2013;Lin et al, 2018;Chandrasekaran et al, 2021;Berndt et al, 2007), which in turn causes a conformational change in the CTT, allowing it to undock and bind the core oscillator protein Timeless (TIM; Chandrasekaran et al, 2021;Lin et al, 2022). The interaction between CRY and TIM then signals for the recruitment of an E3 ubiquitin ligase, Jetlag, to initiate its degradation down the proteasomal pathway (Peschel et al, 2009;Koh et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

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Room-temperature serial synchrotron crystallography of Drosophila cryptochrome

Schneps¹,

Ganguly²,

Crane³

2022

Acta Cryst Sect D Struct Biol

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Fixed-target serial crystallography allows the high-throughput collection of diffraction data from small crystals at room temperature. This methodology is particularly useful for difficult samples that have sensitivity to radiation damage or intolerance to cryoprotection measures; fixed-target methods also have the added benefit of low sample consumption. Here, this method is applied to the structure determination of the circadian photoreceptor cryptochrome (CRY), previous structures of which have been determined at cryogenic temperature. In determining the structure, several data-filtering strategies were tested for combining observations from the hundreds of crystals that contributed to the final data set. Removing data sets based on the average correlation coefficient among equivalent reflection intensities between a given data set and all others was most effective at improving the data quality and maintaining overall completeness. CRYs are light sensors that undergo conformational photoactivation. Comparisons between the cryogenic and room-temperature CRY structures reveal regions of enhanced mobility at room temperature in loops that have functional importance within the CRY family of proteins. The B factors of the room-temperature structure correlate well with those predicted from molecular-dynamics simulations.

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Section: The Overall Structure Is Similar At Cryogenic and Room Tempe...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Room-temperature serial synchrotron crystallography of Drosophila cryptochrome

Schneps¹,

Ganguly²,

Crane³

2022

Acta Cryst Sect D Struct Biol

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“…Protonation of His378 NE2 is likely required for it to interact with the TIM backbone carbonyl and His378 protonation may be responsive to the flavin redox state. The H377L substitution substantially increases the binding affinity to TIM in both light and dark 13 . Surprisingly, His377 does not contact TIM directly but rather anchors the restructured phosphate binding loop by contacting several residues and hydrogen bonding to the Asn253 carbonyl ( Figure 2c ).…”

Section: Figurementioning

confidence: 97%

“…For example, the site of the light-insensitive cryb mutation 39 , an Asp410-to-Arg381 salt bridge proposed from MD simulations to restructure during light activation 40 , appears to break in response to the new conformation of the ribose backbone ( Figure 2d ), in keeping with light driving undocking of the CTT and subsequent rearrangement of the CCM. Two conserved histidine residues within the flavin pocket, His377 and His378, participate in the PL catalytic mechanism and are implicated in CTT release and TIM binding 13, 15, 41 . In the unbound state His378 ND1 hydrogen bonds with O2’ of the FAD ribose, but in the TIM complex, His378 tilts away from the ribose hydroxyl to hydrogen bond to the TIM Leu5 carbonyl with NE2 ( Figures 2b,c and Extended Data Fig.…”

Section: Figurementioning

confidence: 99%

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Cryptochrome-Timeless structure reveals circadian clock timing mechanisms

Lin

Feng

DeOliveira

et al. 2022

Preprint

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Circadian rhythms influence many behaviors and diseases. They arise from oscillations in gene expression caused by repressor proteins that directly inhibit transcription of their own genes. The circadian clock in flies offers a valuable model for studying these processes, wherein Timeless (TIM) plays a critical role in mediating nuclear entry of the transcriptional repressor Period (PER) and the photoreceptor Cryptochrome (CRY) entrains the clock by triggering TIM degradation in light. The cryo-EM structure of the CRY:TIM complex reveals how a light-sensing cryptochrome recognizes its target. CRY engages a continuous core of N-terminal TIM armadillo (ARM) repeats, resembling how photolyases recognize damaged DNA, and binds a C-terminal TIM helix reminiscent of the interactions between light-insensitive CRYs and their partners in mammals. The structure highlights how the CRY flavin cofactor undergoes substantial conformational changes that couple to large-scale rearrangements at the molecular interface, and how a phosphorylated segment in TIM impacts clock period by regulating the binding of importin-α and the nuclear import of TIM and PER. Moreover, the structure reveals that the TIM N-terminus inserts into the restructured CRY pocket to replace the autoinhibitory C-terminal tail released by light, thereby explaining how the LS-TIM polymorphism adapts flies to different climates.

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