pH-dependence of signaling-state formation in Drosophila cryptochrome

Einholz, Christopher; Nohr, Daniel; Rodriguez, Ryan; Topitsch, Annika; Kern, Maria; Goldmann, Jacqueline; Chileshe, Emma; Okasha, Moustafa; Weber, Stefan; Schleicher, Erik

doi:10.1016/j.abb.2021.108787

Cited by 5 publications

(5 citation statements)

References 46 publications

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“…However, CRYD binds to TIM more tightly than lightstate CRY (with a K D in the 1-mM range), indicating that for the WT protein, the lower light-state affinity may be due to incomplete light activation. A contributing factor to incomplete conversion may be that the pH environment of the cell converts some of the flavin ASQ to the NSQ (Einholz et al, 2021), thereby reducing the amount of CRY bound to TIM in light. Furthermore, some apo CRY during overexpression (which we estimate could be as high as 25%) will not contribute to TIM binding but will register in the free sample.…”

Section: Discussionmentioning

confidence: 99%

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

Lin

Schneps²,

Chandrasekaran³

et al. 2022

Structure

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Section: Discussionmentioning

confidence: 99%

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

Lin

Schneps²,

Chandrasekaran³

et al. 2022

Structure

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“…We propose that these changes in the H-bonding network surrounding the FAD trigger changes in H-bonding at the N(5)H and N(3)H that we detect with the rR spectroscopy. Previously, we proposed that these changes resulted in an increase in FADH − /FADH• reduction potential and, consequently, to a ~ 2-fold decrease in an electron transfer rate and ~ 10-fold stabilization of FADH − [28,44]. Such changes are insufficient to fully explain the order in magnitude change in the rate of FADH• oxidation in VcCRY-1 upon substrate-binding.…”

Section: Discussionmentioning

confidence: 94%

“…In the plant CRY subfamily with an FAD ox dark state and FADH• signaling state, it is an aspartate that may donate the proton to the FAD N(5) to form FADH• from FAD• − along with destabilizing a negatively charged FADH − [23][24][25]. The proximal amino acid in the animal CRY subfamily is a cysteine which appears to allow formation of an FAD•signaling state from the FAD ox dark state, though this debate has not been settled [26][27][28]. The CRY-DASH subfamily has an asparagine, just like PL [29], which raises the question how CRY-DASH accesses the presumptive FAD ox dark-state for its signaling function, if that is a requirement, and how it returns back to FADH• and FADH − for its DNA-repair function.…”

Section: Introductionmentioning

confidence: 99%

Investigation of the pH-dependence of the oxidation of FAD in VcCRY-1, a member of the cryptochrome-DASH family

Gindt

Connolly

Haar

et al. 2021

Photochem Photobiol Sci

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Vibrio cholerae cryptochrome-1 (VcCRY-1) is a member of the cryptochrome DASH family. The flavoprotein appears to use blue light both for repair of cyclobutane pyrimidine dimers (CPDs) on DNA and signal transduction. Earlier, we found that it was almost impossible to oxidize the FADH• state upon binding to a CPD, and, in the absence of substrate, the rate of FADH• oxidation was much larger at high pH (Gindt et al. in Biochemistry 54:2802-2805. Here, we present the pH-dependence of the oxidation of FADH• by ferricyanide, which revealed a switch between slow and fast oxidation with a pK a ≈ 7.0. Stopped-flow mixing was used to measure the oxidation of FADH − to FADH• at pH 6.7 and 7.5. Substrate binding was required to slow down this oxidation such that it could be measured with stopped flow, but there was only a small effect of pH. In addition, resonance Raman measurements of FADH• in VcCRY-1 at pH 6.5 and 7.5 were performed to probe for structural changes near the FAD cofactor related to the observed changes in rate of FADH• oxidation. Only substrate binding seemed to induce a change near the FAD cofactor that may relate to the change in oxidation kinetics. The pH-effect on the FADH• oxidation rate, which is rate-limited by the proton acceptor, does not seem to be due to a protein structural change near the FAD cofactor. Instead, a conserved glutamate in CRY-DASH may control the deprotonation of FADH• and give rise to the pH-effect.

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“…However, CRYΔ binds to TIM more tightly than light-state CRY (with a KD in the 1 µM range), indicating that for the WT protein, the lower light-state affinity may be due to incomplete light-activation. A contributing factor to incomplete conversion may be that the pH environment of the cell converts some of the flavin ASQ to the NSQ (Einholz et al, 2021), thereby reducing the amount of CRY bound to TIM in light.…”

Section: Discussionmentioning

confidence: 99%

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

Lin

Schneps

Chandrasekaran

et al. 2021

Preprint

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Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light and triggering its degradation. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM binding. A generally-applicable Select Western-blot-Free Tagged-protein Interaction (SWFTI) assay enables quantification of CRY binding to TIM in dark and light. The assay is utilized 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 bind TIM constitutively, whereas those incapable of photoreduction bind TIM weakly. In response to flavin redox state, two conserved histidine residues contribute to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify protein-protein interactions and photoreceptor targeting.

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pH-dependence of signaling-state formation in Drosophila cryptochrome

Cited by 5 publications

References 46 publications

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

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

Investigation of the pH-dependence of the oxidation of FAD in VcCRY-1, a member of the cryptochrome-DASH family

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

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