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

Pei, Li; Cheng, Huaqiang; Kumar, Vikash; Lupala, Cecylia S.; Li, Xuanxuan; Shi, Yingchen; Ma, Chongjun; Joo, Keehyoung; Lee, Jooyoung; Liu, Haiguang; Tan, Yan‐Wen

doi:10.1038/s42003-022-04054-9

Cited by 6 publications

(7 citation statements)

References 80 publications

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“…As previous ndings indicate CraCRY to take a larger conformation upon illumination, for example the radius of gyration (Rg) increases from 33.5 Å to 34.1 Å, one may expect this to be re ected in the mass spectra and IM heatmaps, as we observed it before for the PYP mutants, for which we performed proof of principle experiments for comparison (Figure S1-3). 13 Those show an increase in the average charge state as well as the CCS, which both re ect a transformation to a larger conformation. The contradictory behavior of CraCRY upon illumination, i.e.…”

Section: Discussionmentioning

confidence: 95%

Light-triggered conformational changes of an animal-like cryptochrome tracked by native time-resolved IMS-MS

Morgner,

Zangl,

Soravia

et al. 2023

Preprint

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Cryptochromes (CRYs) belong to the class of blue light photoreceptors and are responsible for various light-triggered functions in the circadian rhythm upon excitation of their inbuilt flavin cofactor. They are functionally distinct to the evolutionarily related photolyases, which mediate light-driven repair of UV induced DNA damages. Despite these functional differences they share a high degree of structural and sequence homology. A hallmark of cryptochromes is their flexible carboxyl-terminal extension (CTE), whose structure and function as well as the details of its interaction with the photolyase homology region (PHR) are not yet fully understood and differs among different cryptochromes types. In this study we investigate the animal-like cryptochrome from Chlamydomonas reinhardtii (CraCRY). Here, we focus on the highly conserved C-terminal domain harboring the FAD chromophore, to study the effect of single mutations on the structural transition of the C-terminal helix α22 and the attached CTE upon lit-state formation. By coupling a high-power LED, mounted in the source region to an ion mobility mass spectrometer, 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.

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Section: Discussionmentioning

confidence: 95%

Light-triggered conformational changes of an animal-like cryptochrome tracked by native time-resolved IMS-MS

Morgner,

Zangl,

Soravia

et al. 2023

Preprint

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show abstract

“…As previous findings indicate Cr aCRY to take a larger conformation upon illumination, for example the radius of gyration (Rg) increases from 33.5 to 34.1 Å, one may expect this to be reflected in the mass spectra and IM heatmaps, as we observed it before for the PYP mutants, for which we performed proof of principle experiments for comparison (Figure ). Those show an increase in the average charge state as well as the CCS, which both reflect a transformation to a larger conformation. The contradictory behavior of Cr aCRY upon illumination, i.e., compaction in the lit state vs decompaction, when comparing our MS-based approach and smFRET or SAXS, may be a result of the different environments for assessing the light-triggered conformations.…”

Section: Discussionmentioning

confidence: 99%

“…To decipher the structural interaction and the conformational transition upon illumination, structure determining methods are needed that are capable of handling the flexibility of the CTE, where high-resolution methods like NMR or cryo-EM are limited. So far, X-ray crystallography, HDX-MS, or smFRET have been employed to elucidate the conformational change of Cr aCRY upon blue-light irradiation, where some conformational change of the flexible CTE and/or helix α22 due to illumination has been observed. ,, Although HDX-MS can be employed to characterize structural changes in a time-resolved manner, it suffers the ability to analyze directly the kinetics of structural changes, especially in the second and subsecond time regime . Factors affecting the detachment of CTE and the respective time scales of these structural transitions have not yet been comprehensively investigated.…”

Section: Introductionmentioning

confidence: 99%

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

Zangl,

Soravia,

Saft

et al. 2024

J. Am. Chem. Soc.

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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.

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“…Indeed, some studies have suggested that Cr aCRY adopts the red-light-absorbing FADH · state as its resting state 26 , 36 , 37 . However, a recent study showed that the conversion of FAD ox into FADH · turned on the interaction between Cr aCRY and a partner protein, ROC15 (Rhythm of Chloroplast 15), to synchronize the circadian clock with light 38 . Considering these paradoxical results, the resting and signaling states of FAD required for Cr aCRY to perform CRY function remain unclear.…”

Section: Discussionmentioning

confidence: 99%

A deazariboflavin chromophore kinetically stabilizes reduced FAD state in a bifunctional cryptochrome

Hosokawa,

Morita,

Nakamura

et al. 2023

Sci Rep

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An animal-like cryptochrome derived from Chlamydomonas reinhardtii (CraCRY) is a bifunctional flavoenzyme harboring flavin adenine dinucleotide (FAD) as a photoreceptive/catalytic center and functions both in the regulation of gene transcription and the repair of UV-induced DNA lesions in a light-dependent manner, using different FAD redox states. To address how CraCRY stabilizes the physiologically relevant redox state of FAD, we investigated the thermodynamic and kinetic stability of the two-electron reduced anionic FAD state (FADH−) in CraCRY and related (6–4) photolyases. The thermodynamic stability of FADH− remained almost the same compared to that of all tested proteins. However, the kinetic stability of FADH− varied remarkably depending on the local structure of the secondary pocket, where an auxiliary chromophore, 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF), can be accommodated. The observed effect of 8-HDF uptake on the enhancement of the kinetic stability of FADH− suggests an essential role of 8-HDF in the bifunctionality of CraCRY.

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

Cited by 6 publications

References 80 publications

Light-triggered conformational changes of an animal-like cryptochrome tracked by native time-resolved IMS-MS

Light-triggered conformational changes of an animal-like cryptochrome tracked by native time-resolved IMS-MS

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

A deazariboflavin chromophore kinetically stabilizes reduced FAD state in a bifunctional cryptochrome

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