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

Lin, Changfan and Schneps, Connor M. and Chandrasekaran, Siddarth and Ganguly, Abir and Crane, Brian R. (2022) Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome. Structure, 30 (6). 851-861.e5. ISSN 0969-2126. PMCID PMC9201872. doi:10.1016/j.str.2022.03.010.

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Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM recognition. We present a generally applicable select western-blot-free tagged-protein interaction (SWFTI) assay that allowed the quantification of CRY binding to TIM in dark and light. The assay was used to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bound TIM constitutively, whereas those incapable of photoreduction bound TIM weakly. In response to the flavin redox state, two conserved histidine residues contributed to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify the interactions of difficult-to-produce proteins.

Item Type:Article
Related URLs:
URLURL TypeDescription Information CentralArticle Paper
Lin, Changfan0000-0002-8119-9845
Chandrasekaran, Siddarth0000-0003-2990-7397
Ganguly, Abir0000-0002-0630-1109
Crane, Brian R.0000-0001-8234-9991
Additional Information:Published by Elsevier Under a Creative Commons license - Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Received 18 October 2021, Revised 24 January 2022, Accepted 15 March 2022, Available online 8 April 2022, Version of Record 2 June 2022. This work was financially supported by NIH grant R35GM122535 (B.R.C.). ESR measurements were carried out at ACERT which is supported by NIH/NIGMS awards P41 GM103521 and 1S1 0OD021543. The Sortase A plasmid was a gift from William DeGrado at UCSF. We thank Cristina DeOliveira for help with figure preparation. We also thank Robert Dunleavy for his help with ESR data acquisition. Author contributions. C.L., C.M.S., and B.R.C. designed research; and C.L., C.M.S., S.C., A.G. and B.R.C. performed research; C.L., C.M.S., A.G., S.C. and B.R.C. analyzed data; C.L., C.M.S., and B.R.C. wrote the manuscript with contributions from all authors. Data and code availability. Further data for this manuscript are available in the supplemental information document. All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. The authors declare no competing interests.
Funding AgencyGrant Number
NIHP41 GM103521
NIH1S1 0OD021543
Subject Keywords:circadian clock; photoreception; flavoprotein; protein-protein interactions; western blot; affinity assay; conformational change; protein dynamics; electron-spin resonance spectroscopy
Issue or Number:6
PubMed Central ID:PMC9201872
Record Number:CaltechAUTHORS:20220705-346618000
Persistent URL:
Official Citation:Changfan Lin, Connor M. Schneps, Siddarth Chandrasekaran, Abir Ganguly, Brian R. Crane, Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome, Structure, Volume 30, Issue 6, 2022, Pages 851-861.e5, ISSN 0969-2126,
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:115333
Deposited By: George Porter
Deposited On:07 Jul 2022 23:59
Last Modified:15 Jul 2022 17:01

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