Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome.
affinity assay
circadian clock
conformational change
electron-spin resonance spectroscopy
flavoprotein
photoreception
protein dynamics
protein-protein interactions
western blot
Journal
Structure (London, England : 1993)
ISSN: 1878-4186
Titre abrégé: Structure
Pays: United States
ID NLM: 101087697
Informations de publication
Date de publication:
02 06 2022
02 06 2022
Historique:
received:
18
10
2021
revised:
24
01
2022
accepted:
15
03
2022
pubmed:
10
4
2022
medline:
9
6
2022
entrez:
9
4
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 35397203
pii: S0969-2126(22)00091-0
doi: 10.1016/j.str.2022.03.010
pmc: PMC9201872
mid: NIHMS1796888
pii:
doi:
Substances chimiques
Cryptochromes
0
Drosophila Proteins
0
Eye Proteins
0
Flavins
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
851-861.e5Subventions
Organisme : NIGMS NIH HHS
ID : P41 GM103521
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM122535
Pays : United States
Organisme : NIH HHS
ID : S10 OD021543
Pays : United States
Informations de copyright
Published by Elsevier Ltd.
Déclaration de conflit d'intérêts
Declaration of interests The authors declare no competing interests.
Références
Expert Rev Proteomics. 2014 Feb;11(1):107-20
pubmed: 24393018
Chem Biol. 2008 Feb;15(2):128-36
pubmed: 18291317
Methods Enzymol. 2015;563:531-67
pubmed: 26478498
Expert Rev Proteomics. 2014 Oct;11(5):549-60
pubmed: 25059473
Commun Biol. 2021 Feb 26;4(1):249
pubmed: 33637846
Proc Natl Acad Sci U S A. 2016 Sep 6;113(36):10073-8
pubmed: 27551082
J Mol Biol. 2009 Feb 6;385(5):1511-8
pubmed: 19027027
Sci Adv. 2021 Jun 18;7(25):
pubmed: 34144991
Science. 2007 Jun 29;316(5833):1898-900
pubmed: 17600216
Nature. 2013 Mar 21;495(7441):E3-4
pubmed: 23518567
J Phys Chem B. 2016 Aug 4;120(30):7360-70
pubmed: 27404115
Proc Natl Acad Sci U S A. 2018 Apr 10;115(15):3822-3827
pubmed: 29581265
Annu Rev Biochem. 2014;83:191-219
pubmed: 24905781
Nat Chem Biol. 2020 Jun;16(6):676-685
pubmed: 32231341
Arch Biochem Biophys. 2021 Mar 30;700:108787
pubmed: 33545100
Nat Biotechnol. 2003 Jan;21(1):86-9
pubmed: 12469133
J Biol Chem. 2001 Mar 30;276(13):10103-9
pubmed: 11124949
Biochem Biophys Res Commun. 2007 Apr 6;355(2):531-7
pubmed: 17306225
Cell Chem Biol. 2020 Sep 17;27(9):1192-1198.e5
pubmed: 32502390
Int J Proteomics. 2014;2014:147648
pubmed: 24693427
Photochem Photobiol. 2017 Jan;93(1):104-111
pubmed: 27864885
Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):4980-5
pubmed: 23479607
Sci Adv. 2019 Jul 17;5(7):eaaw1531
pubmed: 31328161
J Chem Inf Model. 2018 Oct 22;58(10):2043-2050
pubmed: 30199633
Cold Spring Harb Symp Quant Biol. 2007;72:119-31
pubmed: 18419269
Curr Biol. 2009 Feb 10;19(3):241-7
pubmed: 19185492
Annu Rev Plant Biol. 2011;62:335-64
pubmed: 21526969
J Phys Chem A. 2019 Jan 10;123(1):359-370
pubmed: 30525624
J Biol Chem. 2014 Feb 21;289(8):4634-42
pubmed: 24379403
J Chem Theory Comput. 2012 May 8;8(5):1542-1555
pubmed: 22582031
J Biol Chem. 2007 Feb 16;282(7):4738-4747
pubmed: 17164245
J Biol Rhythms. 2020 Feb;35(1):16-27
pubmed: 31599203
Anal Biochem. 2013 Sep 15;440(2):186-8
pubmed: 23747530
Cell. 1998 Nov 25;95(5):669-79
pubmed: 9845369
Science. 2006 Jun 23;312(5781):1809-12
pubmed: 16794082
Science. 2004 Jun 4;304(5676):1503-6
pubmed: 15178801
Anal Biochem. 2020 Mar 15;593:113608
pubmed: 32007473
Chem Rev. 2003 Jun;103(6):2203-37
pubmed: 12797829
J Mol Recognit. 2014 Jul;27(7):458-69
pubmed: 24895278
Nat Neurosci. 2004 Aug;7(8):834-40
pubmed: 15258584
Cell Chem Biol. 2020 Sep 17;27(9):1109-1111
pubmed: 32946755
Nat Chem Biol. 2014 Oct;10(10):801-9
pubmed: 25229449
Photochem Photobiol. 2017 Jan;93(1):15-25
pubmed: 27925276
Biomed Res Int. 2019 Jan 21;2019:5214821
pubmed: 30800670
Nature. 2011 Nov 13;480(7377):396-9
pubmed: 22080955
Proc Natl Acad Sci U S A. 2015 Feb 17;112(7):2245-50
pubmed: 25646452
Biochemistry. 2016 Sep 20;55(37):5264-71
pubmed: 27529180
Cell Commun Signal. 2020 Nov 16;18(1):182
pubmed: 33198762
Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):20455-60
pubmed: 24297896
Nat Methods. 2009 May;6(5):343-5
pubmed: 19363495