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Published June 2, 2022 | Published + Submitted + Supplemental Material
Journal Article Open

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

Abstract

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.

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.

Attached Files

Published - 1-s2.0-S0969212622000910-main.pdf

Submitted - 2021.09.10.459772v1.full.pdf

Supplemental Material - 1-s2.0-S0969212622000910-mmc1.pdf

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Additional details

Created:
August 22, 2023
Modified:
October 23, 2023