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The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm

Open AccessPublished:October 07, 2020DOI:https://doi.org/10.1074/jbc.RA120.014333
      Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box–driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare CRY1 SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1−/−Cry2−/− double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared with CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.
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      ). PHR and C-terminal regions of the CRYs are required to maintain rhythmicity and amplitude of circadian rhythm, respectively (
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      ).
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      ). Crystal structure of CRY2 PHR-PER2 C-terminal CRY-binding domain (CBD) revealed an unusual binding mode of PER2-CBD onto CRY2 (
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      ). The N-terminal half of PER2-CBD reaches the rim of secondary pocket of the CRY2-PHR. This interaction allows the serine loop, located adjacent to the secondary pocket, to adapt different conformations. A recent study indicates that PER2 remodels the serine loop of CRY2 and, in turn, enhances its affinity toward BMAL1/CLOCK (
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      ). In the same study, replacement of amino acid residues in the serine loop of CRY1 reduced its affinity to BMAL1/CLOCK. However, communication and allosteric regulation between primary and secondary pockets of CRY1 have not been reported so far.
      Here, we characterized three SNPs (p.Gly144Val, p.Arg293His, and p.Arg348Cys) identified from 1000 Genomes project and Ensembl databases. Among them, analysis of the p.Arg293His CRY1 variant indicated that it had a longer t1/2 and weaker repression activity and caused shorter period of circadian rhythm. Further biochemical analysis showed that this variant had a weaker affinity toward the BMAL1/CLOCK complex in the absence of PER2. Interestingly, a comparison of CRY2 and the p.Arg293His CRY1 variant showed very similar molecular characteristics in terms of t1/2 and binding properties to BMAL1/CLOCK. However, in rescue assays using Cry1−/−Cry2−/− mouse embryonic fibroblasts CRY2 and p.Arg293His CRY1 showed some differences. The period length of rhythm observed in p.Arg293His CRY1 rescue cell lines were shorter than WT CRY1, but longer than CRY2. To understand all these changes mediated by Arg-293 within the structural context of CRY1, we performed MD simulations. The MD studies of both mutant and WT CRY1 suggest that Arg-293 is involved in allosteric regulation of CLOCK binding by regulating the dynamicity of the serine loop adjacent to the secondary pocket.
      Collectively, this study provides new insights into the circadian clock mechanism. In particular, our study suggests potential new targets for drug discovery by identifying novel amino acids affecting the repressor and binding activities of the CRY1 and the possible allosteric regulation in the CRY1 secondary pocket.

      Results

      Selection of CRY1 SNPs

      Several SNPs with either low or high frequencies are associated with different types of diseases. However, the functional significance of rare missense mutations deduced from large-scale genomic studies and how these mutations affect protein function are largely unknown. Because of that, we investigated the effect of missense variants of the human CRY1 gene. The SNPs for CRY1 were selected from the 1000 Genomes project and Ensembl databases (
      • Auton A.
      • Brooks L.D.
      • Durbin R.M.
      • Garrison E.P.
      • Kang H.M.
      • Korbel J.O.
      • Marchini J.L.
      • McCarthy S.
      • McVean G.A.
      • Abecasis G.R.
      1000 Genomes Project Consortium
      A global reference for human genetic variation.
      ,
      • Zerbino D.R.
      • Achuthan P.
      • Akanni W.
      • Amode M.R.
      • Barrell D.
      • Bhai J.
      • Billis K.
      • Cummins C.
      • Gall A.
      • Girón C.G.
      • Gil L.
      • Gordon L.
      • Haggerty L.
      • Haskell E.
      • Hourlier T.
      • et al.
      Ensembl 2018.
      ) based on in silico analyses and their location within the CRY1 structure. Among 12,000 CRY1 SNPs, a majority of them are located within noncoding regions. Of the SNPs reported within the CRY1 coding region (116 missense mutations are detected), we have focused on missense mutations affecting the PHR domain of the CRY1, which is crucial for its function (
      • Khan S.K.
      • Xu H.Y.
      • Ukai-Tadenuma M.
      • Burton B.
      • Wang Y.M.
      • Ueda H.R.
      • Liu A.C.
      Identification of a novel cryptochrome differentiating domain required for feedback repression in circadian clock function.
      ). Three of these variants predicted to be detrimental to protein structure or function were selected based on their SIFT, PolyPhen, and Provean scores (Table 1). These variants were interpreted as “disease-causing” by MutationTaster (
      • Schwarz J.M.
      • Cooper D.N.
      • Schuelke M.
      • Seelow D.
      MutationTaster2: Mutation prediction for the deep-sequencing age.
      ). Additionally, VarSome-Verdict and ClinVar were used to understand the clinical effects of these variants. Although these variants were classified as “variant of uncertain significance-VUS”, and all of them meet two pathogenic American College of Medical Genetics and Genomics criteria (guidelines for interpretation of sequence variants) (
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • Grody W.W.
      • Hegde M.
      • Lyon E.
      • Spector E.
      • Voelkerding K.
      • Rehm H.L.
      ACMG Laboratory Quality Assurance Committee
      Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ). None of these variants were reported in ClinVar. The p.Arg293His and p.Arg348Cys CRY1 variants were detected as heterozygous; the genotype of the p.Gly144Val was not reported in Ensembl. To analyze the degree of conservation of these SNPs, we performed multiple sequence alignment (Fig. 1A). The Arg-293 in CRY1 is conserved in all CRY1, whereas Gly-144 is conserved in all CRY1 proteins with the exception of mosquito CRY1. Finally, Arg-348 is conserved in CRY1 from most organisms, but not in CRY1 from sponge, mosquito, housefly, green sea turtle, blowfly, and stony coral. WT amino acids in all three SNPs are highly conserved in the CRY1 across different organisms, which suggests a potential role in protein function (Fig. 1A). Thus, we aimed to investigate the effects of these variants at the molecular level. To do that, we generated mutant constructs of mouse Cry1 (mCry1) by site-directed mutagenesis, and these variants were subjected to further functional analysis.
      Table 1The clinical features of selected CRY1 SNPs
      dbSNPPosition in CDS (NM_004075.4)Position in protein (NP_004066.1)SIFT/PolyPhen/ProveanACMGgnomAD exomClinVar
      rs78310335c.431G>Tp.Gly144Val0/0.999/damagingVUS (PM2, PP3)not detected
      rs772585429c.878G>Ap.Arg293His0/1/damagingVUS (PM2, PP3)ƒ = 0.00000797
      rs749506981c.1042C>Tp.Arg348Cys0/0.989/damagingVUS (PM2, PP3)not detected
      Figure thumbnail gr1
      Figure 1Three rare SNPs affect highly conserved amino acid residues in CRY1. A, alignments of selected each class of the cryptochrome/photolyase family proteins from the phylogenetic tree recently constructed by Kavakli et al. (
      • Kavakli I.H.
      • Ozturk N.
      • Gul S.
      DNA repair by photolyases.
      ). Human CRY1 was indicated highlighted in gray color and in bold. Protein accession number organisms are as follows: Amphimedon queenslandica CRY1 (accession number: XP_003386582.1), Danaus plexippus Cry1 (accession number: AAX58599.1), Helicoverpa armigera CRY1 (accession number: XP_021184243.1), Anopheles gambiae CRY1 (accession number: NP_034093), Drosophila melanogaster Cry (accession number: BAA05042.1), Lucilia cuprina CRY1 (accession number: XP_023306440.1), Musca domestica CRY1 (accession number: XP_005178207), Drosophila melanogaster (6-4)PHR (accession number: NP_001260633.1), Acropora millepora CRY1 (accession number: ABP97098.1), Danio rerio Cry1a (accession number: BAA96846.1), Xenopus laevis CRY1 (accession number: NP_001081129.1), Rattus norvegicus CRY1 (accession number: NP_942045.2), Homosapiens CRY1 (accession number: NP_004066.1 ×), Mus musculus CRY1 (accession number: NP_031797.1), Sylvia borin CRY1b (accession number: ABH03083.1), Gekko japonicas CRY1 (accession number: XP_015283074.1), Gallus gallus Cry1 (accession number: NP_989576.1), Alligator sinensis CRY1 (accession number: XP_006024339.1), Tribolium castaneum CRY (accession number: NP_001076794.1), Escherichia coli PHR CPD (accession number: WP_032176081.1), Arabidopsis thaliana CRY1 (accession number: NP_567341.1), and Solanum lycopersicum CRY1 (accession number: AAF72555.1). B, positions of SNPs were visualized in the mCRY1 (PDB ID: 4K0R) structure using Pymol (RRID:SCR_000305). The mCry1 protein structure is shown as a ribbon diagram (left) or surface (right), and the mutant residues are shown as sticks. C, surface representation of CRY1 residues in the FAD binding pocket and in the secondary pocket were colored red and cyan, respectively, as described in Rosensweig et al. (
      • Rosensweig C.
      • Reynolds K.A.
      • Gao P.
      • Laothamatas I.
      • Shan Y.L.
      • Ranganathan R.
      • Takahashi J.S.
      • Green C.B.
      An evolutionary hotspot defines functional differences between CRYPTOCHROMES.
      ). Gly-144 and Arg-348 are exposed to solvent; Arg-293 is located in the FAD binding pocket facing the secondary pocket. D, Arg-293 is contacting FBXL3. CRY1 structure was superimposed to CRY2-FBXL3 complex in PDB ID 4I6J.

      The effect of SNPs on CRY1 t1/2 and molecular clock function

      The functional characteristics of the selected rare missense variations were studied in detail using biochemical and cell-based assays. Mapping of SNPs onto the crystal structure of CRY1 indicated that these SNPs are located on functionally important domains. Gly-144 and Arg-348 are both exposed to solvent and are located on the loop between helices α5 and α6, or in helix α14, respectively (Fig. 1B). Arg-293 is located within the FAD binding pocket and FBXL3 interaction site which is adjacent to the secondary pocket (Fig. 1, B and C). Because FBXL3 is the E3 ligase responsible for ubiquitination of CRY1, mutations in this region of FBXL3 might change the stability of CRY1 (Fig. 1D). We, therefore, investigated the effect of these mutations on the t1/2 of CRY1 by monitoring the degradation of CRY1 fused to LUC (CRY1::LUC) at the C-terminal. After generating Cry1 variants, plasmids were transiently transfected into HEK293T cells, which were then treated with cycloheximide to inhibit protein synthesis. Luminescence was monitored to determine the t1/2 of the proteins. Stability of the p.Arg348Cys CRY1 and p.Gly144Val CRY1 variants were found to be similar to WT CRY1 (Fig. 2A). However, the t1/2 of the p.Arg293His CRY1 variant was significantly longer than that of WT CRY1 and shorter than luciferase. We further confirm this result with biochemical assays. After transfection of HEK293T cells with proper plasmids, cells were treated with cycloheximide and harvested at the 4th and 8th h. Analysis of samples with Western blotting indicated that p.Arg293His CRY1 is indeed more stable than the WT CRY1 (Fig. 2A, right panel). These results suggested that although mutations distant from FBXL3 interaction sites did not change the stability of CRY1, mutation in the binding region of FBXL3 (i.e. p.Arg293His) increased the t1/2 of CRY1 probably because of the attenuated interaction between CRY1 and FBXL3.
      Figure thumbnail gr2
      Figure 2t1/2 and rescue analysis of the WT and CRY1 variants. A, the half-lives of WT and variant CRY1 were determined by monitoring CRY1::LUC degradation. Half-lives were calculated using a one-phase exponential decay function. Average t1/2 values ± S.E. from three independent experiments (with quadruplicate samples) relative to WT CRY1::LUC were reported. Statistical significance was determined as in (B) (G144V, p = 0.427; R293H, p = 0.013; R348C, p = 0.227). Statistical analysis for Western blot was performed with student's t-test *, (p < 0.0297). B, WT or variant CRY rescued circadian rhythms in Cry1−/−Cry2−/− MEF cells with varying effects on period and amplitude. DKO MEF cells transiently transfected with pGL3-Per2-dLuc and Cry rescue plasmids (WT or variant). After 72 h, cells were synchronized with dexamethasone (0.1 μm) and luminescence values were recorded for 5 days. CRY1 rhythm was representative bioluminescence of four independent experiments with eight plates. The circadian period from all plates were determined. Statistical analysis was performed using an unpaired t test with a Welch's correction (***, p < 0.0001; **, p < 0.005; *, p < 0.05; G144V, p < 0.0001; R293H, p < 0.0001; R348C, p < 0.0044).
      We next determined the effect of these variants on circadian clock function. To do this, we employed a complementation assay using Cry1−/−Cry2−/− mouse embryonic fibroblast cells (DKO-MEF). Plasmid constructs carrying WT Cry1 were shown to rescue circadian rhythms in DKO-MEF when expressed under the control of mCry1 promoter containing E/E′-box and D-box elements and the ROR/REV-ERB-binding element in the first intron (
      • Ukai-Tadenuma M.
      • Yamada R.G.
      • Xu H.Y.
      • Ripperger J.A.
      • Liu A.C.
      • Ueda H.R.
      Delay in feedback repression by Cryptochrome 1 is required for circadian clock function.
      ). Thus, we introduced the p.Gly144Val, p.Arg293His, and p.Arg348Cys variants into the pMU2-Cry plasmid. The Cry expression vector carrying either the variants or WT Cry1 and the mPer2-dLuc reporter plasmid were transiently expressed in DKO-MEF cells. We monitored bioluminescence for 5 days. Although WT and variants were able to restore the circadian rhythm, they had varying effects on period length. The period length for WT CRY1 was calculated as 26.3 h (Fig. 2B). The p.Gly144Val and p.Arg348Cys CRY1 variants increased the period length to 27.8 and 27 h, respectively. Despite the longer t1/2 of the p.Arg293His CRY1 variant, the mutant caused a significantly shorter period length (23.8 h) and displayed weaker repressor activity based on higher luminescence intensities compared with WT CRY1. Because rhythm is generated as a result of interaction between core clock proteins, e.g. CRY1, PER2, BMAL1, and CLOCK, this intriguing observation on p.Arg293His mutant led us to analyze binding properties of these mutants to the core clock proteins.

      p.Arg293His CRY1 binding to BMAL1/CLOCK is differentially disrupted

      To assess binding of each CRY1 variants to CLOCK and BMAL1, HEK293T cells were transiently transfected with expression plasmids coding for CLOCK, Bmal1, and either WT or Cry1 variants. The CRY1-myc-His (variants or WT)–containing protein complexes were then precipitated using Ni-NTA agarose, and the samples were probed for the presence of CLOCK and BMAL1 by Western blot analysis as described in Ref.
      • Rosensweig C.
      • Reynolds K.A.
      • Gao P.
      • Laothamatas I.
      • Shan Y.L.
      • Ranganathan R.
      • Takahashi J.S.
      • Green C.B.
      An evolutionary hotspot defines functional differences between CRYPTOCHROMES.
      . To assess the binding of CLOCK properly, comparable amount of the CRYs were used in pulldown assay. The p.Gly144Val and p.Arg348Cys CRY1 variants exhibited similar affinities to BMAL1/CLOCK compared with WT CRY1, whereas the p.Arg293His CRY1 variant exhibited significantly less affinity to BMAL1/CLOCK (Fig. 3A). A similar experiment was performed in the presence and absence of PER2 to investigate the possibility that PER2 is required for proper binding. All three CRY1 variants and WT CRY1 bound similar amounts of PER2. Nevertheless, the p.Arg293His CRY1 variant was to bind less BMAL1/CLOCK in the absence of PER2 (Fig. 3B). Biochemical binding analysis showed that reduced repression by the p.Arg293His variant of CRY1 is most likely caused by attenuated binding to the BMAL1/CLOCK dimer. However, the p.Gly144Val and p.Arg348Cys variants retained binding activity to the BMAL1/CLOCK dimer. Overall, the p.Arg293His CRY1 variant had longer t1/2, rescued circadian rhythms with shorter period in a weakly repressed state in DKO-MEF cells (Fig. 3B) and exhibited reduced affinity for the BMAL1/CLOCK dimer even in the presence of PER2 (Fig. 3). These features of the p.Arg293His CRY1 variant reported here are very similar to those reported for CRY2 (
      • Khan S.K.
      • Xu H.Y.
      • Ukai-Tadenuma M.
      • Burton B.
      • Wang Y.M.
      • Ueda H.R.
      • Liu A.C.
      Identification of a novel cryptochrome differentiating domain required for feedback repression in circadian clock function.
      ,
      • Rosensweig C.
      • Reynolds K.A.
      • Gao P.
      • Laothamatas I.
      • Shan Y.L.
      • Ranganathan R.
      • Takahashi J.S.
      • Green C.B.
      An evolutionary hotspot defines functional differences between CRYPTOCHROMES.
      ), which led us to perform additional studies.
      Figure thumbnail gr3
      Figure 3Pulldown analysis. A and B, physical interaction between WT/p.Arg293His CRY1variant with (A) BMAL1/CLOCK and (B) BMAL1/CLOCK/PER2. HEK293T cells were transfected with plasmids expressing Bmal1-FLAG, CLOCK-FLAG, and WT or variant Cry1-His-Myc. Proteins were isolated with a Ni-NTA agarose resin and analyzed by Western blotting. The blot shown is representative of three independent experiments. Multiple bands in BMAL1 and CLOCK are a result of posttranslational modifications. Results are shown here are representations of three independent experiments.

      p.Arg293His CRY1 variant exhibits similar characteristics to CRY2

      The secondary pocket of CRY1 is critical for its interaction with CLOCK. Subtle changes in this pocket can result in substantial differences in periodicity in cycling cells (
      • Michael A.K.
      • Fribourgh J.L.
      • Chelliah Y.
      • Sandate C.R.
      • Hura G.L.
      • Schneidman-Duhovny D.
      • Tripathi S.M.
      • Takahashi J.S.
      • Partch C.L.
      Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1.
      ,
      • Fribourgh J.L.
      • Srivastava A.
      • Sandate C.R.
      • Michael A.K.
      • Hsu P.L.
      • Rakers C.
      • Nguyen L.T.
      • Torgrimson M.R.
      • Parico G.C.G.
      • Tripathi S.
      • Zheng N.
      • Lander G.C.
      • Hirota T.
      • Tama F.
      • Partch C.L.
      Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing.
      ). For example, differential rhythms driven by CRY1 and CRY2 are the result of these changes (
      • Rosensweig C.
      • Green C.B.
      Periodicity, repression, and the molecular architecture of the mammalian circadian clock.
      ). To directly compare p.Arg293His CRY1 and WT CRY2, we measured their half-lives and effects on circadian rhythms using DKO-MEF cells. Both the pArg293His CRY1 variant and WT CRY2 exhibited similar half-lives (Fig. 4A). It has been shown that CRY2 can rescue circadian rhythms when the pMU2 plasmid with Cry2 cDNA is transfected in DKO-MEFs (
      • Rosensweig C.
      • Reynolds K.A.
      • Gao P.
      • Laothamatas I.
      • Shan Y.L.
      • Ranganathan R.
      • Takahashi J.S.
      • Green C.B.
      An evolutionary hotspot defines functional differences between CRYPTOCHROMES.
      ,
      • Ukai-Tadenuma M.
      • Yamada R.G.
      • Xu H.Y.
      • Ripperger J.A.
      • Liu A.C.
      • Ueda H.R.
      Delay in feedback repression by Cryptochrome 1 is required for circadian clock function.
      ). Therefore, we cloned Cry2 cDNA into the pMU2 plasmid and transfected this construct into DKO-MEF to see whether it rescues circadian rhythms. Compared with CRY1, CRY2 was able to rescue circadian rhythms with a shorter period, which was consistent with a previous study (
      • Rosensweig C.
      • Reynolds K.A.
      • Gao P.
      • Laothamatas I.
      • Shan Y.L.
      • Ranganathan R.
      • Takahashi J.S.
      • Green C.B.
      An evolutionary hotspot defines functional differences between CRYPTOCHROMES.
      ). Our results showed that cells expressing CRY2 had higher overall luminescence intensity than cells expressing CRY1, suggesting higher BMAL1/CLOCK activity, again consistent with a previously published report (
      • Khan S.K.
      • Xu H.Y.
      • Ukai-Tadenuma M.
      • Burton B.
      • Wang Y.M.
      • Ueda H.R.
      • Liu A.C.
      Identification of a novel cryptochrome differentiating domain required for feedback repression in circadian clock function.
      ). Interestingly, cells expressing the p.Arg293His CRY1 or CRY2 had similar luminescence level (Fig. 4B). In the complementation assay, DKO-MEF cells expressing WT CRY1 or CRY2 had circadian periods of 26.2 and 22.9 h, respectively. Cells expressing the p.Arg293His CRY1 variant had a period length of 24.1 h, which is shorter than that of WT CRY1 and longer than CRY2 (Fig. 4B). Finally, the amplitude of the circadian rhythm was higher in cells expressing p.Arg293His CRY1 variant and CRY2 than WT CRY1 (Fig. 4B). To compare the ability of p.Arg293His CRY1 and CRY2 to repress BMAL1/CLOCK transactivation, we used an mPer1-dLuc assay using Cry1−/−Cry2−/− MEF cells. These results indicated that both CRY2 and p.Arg293His CRY1 had comparable levels of repressor activity that was dose-dependent and statistically significantly different from that of WT CRY1 (Fig. 4C). Additionally, binding of WT and p.Arg293His CRY1 and CRY2 to BMAL1, CLOCK, and PER2 were analyzed side by side (Fig. 4D). We performed pulldown assay in the absence and presence of PER2 to assess the binding ability of WT CRY1, CRY2, and p.Arg293His CRY1 to CLOCK. All CRYs tested in this study had comparable binding to CLOCK in the presence of PER2 (Fig. 4D). However, CRY2 and p.Arg293His CRY1 had attenuated binding to CLOCK compared with WT CRY1 in the absence of the PER2.
      Figure thumbnail gr4
      Figure 4Comparison of protein t1/2 and analysis of rescue of CRY1 and CRY2. A, The half-lives of WT, R293H CRY1, and CRY2 were determined as in . Average t1/2 values ± S.E. from three independent experiments (with triplicate samples) relative to WT CRY1::dLUC were reported (left panel). Statistical analysis was performed using an unpaired t test with a Welch's correction (***, p < 0.0001; **, p < 0.005; *, p < 0.05; WT CRY1 versus R293H, p = 0.0226; WT CRY1 versus CRY2, p = 0.0034; R293H CRY1 versus CRY2, p = 0.4854). Degradation of WT CRY1 and R293H CRY1 was analyzed by immunoblot using anti-Myc for CRYs and anti-β-actin for loading controls (right panel). Degradation of LUC was compared with WT and R293H CRY1::LUC. B, pArg293His CRY1 and CRY2 rescued the rhythm similarly, although periods are still significantly different. Rescue assays were performed as described in . Representative rhythms of three independent experiments are shown. Periods and amplitudes of all plates were reported (4-WT, 6-R293H CRY1, and 6-CRY2). Significance analysis was performed using unpaired t test with Welch's correction (***, p < 0.0001; **, p < 0.005; *, p < 0.05; WT CRY1 versus R293H, p < 0.0001; WT CRY1 versus CRY2, p < 0.0001; R293H CRY1 versus CRY2, p < 0.0001). C, repression analysis. Cry1−/−Cry2−/− MEF cells were transfected with plasmids encoding Bmal1 (50 ng), Clock (125 ng), and different amounts of WT Cry1, Cry2, and Cry1R293H, along with the pGL3-mPer1:luc (50 ng) reporter plasmid and pRL-TK Renilla (4 ng) as a transfection control. Comparisons were made between WT CRY1 and CRY1R293H and WT CRY1 and CRY2 (***, p < 0.0001; **, p < 0.005; *, p < 0.05). D, pulldown analysis of WT or pArg293His CRY1, CRY2 with BMAL1, CLOCK, and PER2. HEK293T cells were transfected with plasmids expressing Bmal1-FLAG, CLOCK-FLAG, and Cry-His-Myc. Proteins were pull downed with Ni-NTA agarose resin and analyzed with Western blotting. This blot is the representation of three independent experiments.
      Collectively, our results suggested that the p.Arg293His CRY1 variant possesses similar properties to CRY2 in terms of stability, binding to core clock components, repression activity and rescuing circadian rhythms in DKO-MEF, except p.Arg293His CRY1 generated longer rhythm than CRY2.

      Understanding the role of Arg-293 in CRY1 function

      Although p.Arg293His CRY1 variant exhibits CRY2-like in vitro properties, the conservation of this amino acid residue in all mammalian CRY1s suggests that it might play an important role, such as regulation of CLOCK binding to the secondary pocket of CRY1. To investigate this, we employed MD simulations using CRY1 structural data. Recently, it has been shown that CRYs bind to the CLOCK PASB domain through the secondary pocket, and this is regulated by the serine loop (
      • Fribourgh J.L.
      • Srivastava A.
      • Sandate C.R.
      • Michael A.K.
      • Hsu P.L.
      • Rakers C.
      • Nguyen L.T.
      • Torgrimson M.R.
      • Parico G.C.G.
      • Tripathi S.
      • Zheng N.
      • Lander G.C.
      • Hirota T.
      • Tama F.
      • Partch C.L.
      Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing.
      ). We, therefore, explored possible dynamical differences between WT CRY1 and p.Arg293His CRY1 around the secondary pocket via MD simulations. p.Gly144Val CRY1 and p.Arg348Cys CRY1 structures were also simulated as controls. For this purpose, we utilized mouse CRY1 crystal structure to generate a complete PHR domain of the individual variants. Each protein was sampled for 300 ns, and the root mean square deviation (RMSD) values were determined to show that simulations reached equilibrium (Fig. 5A). To investigate the dynamics of each residue, root mean square fluctuation (RMSF) values were assessed (Fig. 5B). We observed differences in the RMSF values of residues in the serine loop (residues 38–48). When compared with WT CRY1, the residues in p.Arg293His CRY1 simulations showed minimal dynamism/fluctuation (Fig. 5B). Because the serine loop regulates the volume and access to the secondary pocket, we analyzed the volume of the secondary pocket using POVME 3.0 software. Analysis showed that simulations of p.Arg293His CRY1 sampled significantly smaller secondary pocket volume than WT CRY1 (Fig. 5C). Secondary pocket volume of p.Gly144Val CRY1 was comparable to WT CRY1. Interestingly, p.Arg348Cys CRY1 simulation sampled a larger secondary volume pocket. Because secondary pocket volume of WT CRY1 allows CLOCK binding, we do not expect larger pocket volume will affect CLOCK binding to p.Arg348Cys CRY1. In fact, rescue assays (Fig. 2B) and BMAL1/CLOCK binding analysis to CRY1 variants (Fig. 3) confirmed that p.Arg348Cys mutation did not change the binding of CRY1 to CLOCK. To further examine the relative internal dynamics of the loop, we analyzed the distance between the Cα atoms of Gly-43 located on the loop and Phe-105 (Fig. 5, D and E). The average loop distances of CRY1 and p.Arg293His CRY1 were not significantly different. However, the variance of loop distances sampled throughout the simulations were significantly different (Levene's test). To compare the scatter of loop distance and its correlation with the volume of the secondary pocket, we plotted loop distance against the secondary pocket volume (Fig. 5E). Our analysis of secondary pocket volume and loop distance showed that p.Arg293His CRY1 behaved similarly to WT CRY1 (Fig. 5, CE). Despite this similarity between CRY1 and p.Arg293His CRY1 simulations, p.Arg293His CRY1 only sampled a fraction of the space explored by WT CRY1 (Fig. 5E). In line with secondary pocket volume analysis, p.Gly144Val sampled similar loop distance to WT CRY, p.Arg348Cys sampled longer loop distance. These observations in MD simulations implied that the dynamics of the serine loop in CRY1 and p.Arg293His CRY1 are regulated differently. Although the serine loop in WT CRY1 is more dynamic, reduced dynamicity of this loop in p.Arg293His CRY1 might explain the attenuated binding of p.Arg293His CRY1 to CLOCK. To explain this differential regulation on the serine loop in detail, we analyzed possible communication pathways between Arg-293 and the serine loop by using Weighted Implementation of the Suboptimal Paths (WISP) software as described in Ref.
      • Ozdemir E.S.
      • Jang H.
      • Gursoy A.
      • Keskin O.
      • Li Z.
      • Sacks D.B.
      • Nussinov R.
      Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2.
      . The WISP algorithm can calculate both optimal and suboptimal (longer) communication pathways between nodes (residues). Residue 293 was used as a source residue, and several possible residues on the serine loop (Gly-43, Ser-44, Ser-45, Asn-46) were used as a sink to determine promising pairs for communicating pathways. A total of 500 pathways between residues Arg-293 and Gly-43 for each WT and p.Arg293His CRY1 were calculated. The shortest pathway length for p.Arg293His CRY1 was calculated as 3.26 Å. The shortest communicating pathway involves the His-293, Ile-392, Ser-391, Trp-390, Gly-43 residues. However, the length of shortest pathway for WT CRY1 was calculated as 5.19 Å, which involves the Arg-293, Phe-296, Ala-300, Pro-104, Gly-106, Arg-109, Trp-8, Ile-36, Asp-38, Gly-43 residues (Fig. 5, F and G). Not only is the shortest pathway changed, but also the suboptimal pathways sampled longer lengths in p.Arg293His CRY1 (Fig. 5G). Residues involved more than 200 suboptimal paths between Arg-293/His-293 and Gly-43 are Asp-38, Pro-104, Gly-106, Ile-36, Ala-300, Arg-109, Phe-296, Trp-8, and Ala-299 for WT CRY1; Glu-382, Phe-296, Gln-393, and Ile-392 for p.Arg293His CRY1. Both representation of pathways and histogram of pathway lengths showed that the mutation introduced new communicating pathways between His-293 and Gly-43. Taken together, these computational findings suggest that the serine loop is allosterically regulated by residue Arg-293 in CRY1 and reduces the dynamicity of the serine loop, keeping it in a closed conformation. Our search in the ClinVar database revealed that there was no disease phenotype associated with this variant.
      Figure thumbnail gr5
      Figure 5Molecular dynamic simulation studies of mutant and WT CRY1. A, RMSD of CRY1 WT, G144V, R293H, and R348C CRY1 simulations. B, RMSF of CRY1 WT and pArg293His CRY1. Residue numbering is according to CRY1. Residues between two dashed lines belong to the serine loop. C, box plot representation of the secondary pocket volume sampled throughout the simulations. Wilcoxon rank sum test was applied (*, p <0.0001). D, box plot representation of the loop distance produced by measuring the distance between Cα atoms of Gly-43 and Phe-105. Distance between Gly-43(on loop) and Phe-105 was calculated with a custom Python script. The same multi-frame PDB files extracted from the trajectories were used to calculate the secondary pocket volume and loop distances. Statistical analysis between variances of loop distances of CRY1 and CRY1R293H simulations was calculated by the Levene's test (p < 0.001). E, scatter plot of the loop distance against the secondary pocket volume in WT and R293H CRY1 simulations. F, WISP analysis of the 500 pathways for CRY1 WT and CRY1R293H simulations. Red spheres represent the nodes participating in the communication pathway between residues 293 and 43 in CRY1. Each node corresponds to a residue. Residues Arg-293 or His-293 and Gly-43 were shown as black spheres. G, histographic representation of the 500 pathways' lengths calculated by WISP.

      Discussion

      Mammalian CRYs are integral components of the circadian clock mechanism and are strong transcriptional repressors of BMAL1/CLOCK transactivation (
      • Kavakli I.H.
      • Sancar A.
      Circadian photoreception in humans and mice.
      ,
      • Sato T.K.
      • Yamada R.G.
      • Ukai H.
      • Baggs J.E.
      • Miraglia L.J.
      • Kobayashi T.J.
      • Welsh D.K.
      • Kay S.A.
      • Ueda H.R.
      • Hogenesch J.B.
      Feedback repression is required for mammalian circadian clock function.
      ,
      • Ueda H.R.
      • Hayashi S.
      • Chen W.B.
      • Sano M.
      • Machida M.
      • Shigeyoshi Y.
      • Iino M.
      • Hashimoto S.
      System-level identification of transcriptional circuits underlying mammalian circadian clocks.
      ). Several genetic studies have highlighted the relationship between CRY variants and different types of diseases. For example, CRY1 variants have been associated with depression and mood disorders (
      • Hua P.
      • Liu W.
      • Chen D.
      • Zhao Y.
      • Chen L.
      • Zhang N.
      • Wang C.
      • Guo S.
      • Wang L.
      • Xiao H.
      • Kuo S.H.
      Cry1 and Tef gene polymorphisms are associated with major depressive disorder in the Chinese population.
      ,
      • Kovanen L.
      • Donner K.
      • Kaunisto M.
      • Partonen T.
      CRY1, CRY2 and PRKCDBP genetic variants in metabolic syndrome.
      ,
      • Soria V.
      • Martínez-Amorós E.
      • Escaramís G.
      • Valero J.
      • Pérez-Egea R.
      • García C.
      • Gutiérrez-Zotes A.
      • Puigdemont D.
      • Bayés M.
      • Crespo J.M.
      • Martorell L.
      • Vilella E.
      • Labad A.
      • Vallejo J.
      • Pérez V.
      • et al.
      Differential association of circadian genes with mood disorders: CRY1 and NPAS2 are associated with unipolar major depression and CLOCK and VIP with bipolar disorder.
      ), elevated blood pressure and hypertension (
      • Kovanen L.
      • Donner K.
      • Kaunisto M.
      • Partonen T.
      CRY1, CRY2 and PRKCDBP genetic variants in metabolic syndrome.
      ), and insulin resistance (
      • Dashti H.S.
      • Smith C.E.
      • Lee Y.C.
      • Parnell L.D.
      • Lai C.Q.
      • Arnett D.K.
      • Ordovás J.M.
      • Garaulet M.
      CRY1 circadian gene variant interacts with carbohydrate intake for insulin resistance in two independent populations: Mediterranean and North American.
      ,
      • Kovanen L.
      • Donner K.
      • Kaunisto M.
      • Partonen T.
      CRY1 and CRY2 genetic variants in seasonality: A longitudinal and cross-sectional study.
      ). This clinical heterogeneity implies a variety of different functions for CRY1 potentially caused by different functional domains of the protein. Multiple sequence alignment shows that CRYs have variable extended C-terminal domains that range from 30 to 300 amino acids depending on the species (
      • Kavakli I.H.
      • Baris I.
      • Tardu M.
      • Gül Ş.
      • Öner H.
      • Çal S.
      • Bulut S.
      • Yarparvar D.
      • Berkel Ç.
      • Ustaoğlu P.
      • Aydın C.
      The photolyase/cryptochrome family of proteins as DNA repair enzymes and transcriptional repressors.
      ,
      • Partch C.L.
      • Clarkson M.W.
      • Ozgür S.
      • Lee A.L.
      • Sancar A.
      Role of structural plasticity in signal transduction by the cryptochrome blue-light photoreceptor.
      ). N-terminal domain exhibits high homology to photolyases and is called the PHR domain.
      The C terminus of mammalian CRY possesses a highly conserved helix that has a periodic spacing of nonpolar residues common to coiled-coil and is called the coiled-coil helix (
      • Chaves I.
      • Yagita K.
      • Barnhoorn S.
      • Okamura H.
      • van der Horst G.T.J.
      • Tamanini F.
      Functional evolution of the photolyase/cryptochrome protein family: Importance of the C terminus of mammalian CRY1 for circadian core oscillator performance.
      ). In mammals, this region is competitively bound by FBXL3 (
      • Xing W.
      • Busino L.
      • Hinds T.R.
      • Marionni S.T.
      • Saifee N.H.
      • Bush M.F.
      • Pagano M.
      • Zheng N.
      SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket.
      ) and PER2 (
      • Ozber N.
      • Baris I.
      • Tatlici G.
      • Gur I.
      • Kilinc S.
      • Unal E.B.
      • Kavakli I.H.
      Identification of two amino acids in the C-terminal domain of mouse CRY2 essential for PER2 interaction.
      ) to regulate the stability of CRYs, and to tune the circadian clock machinery. The PHR domain essentially consists of two important regions, an α-helical domain (primary pocket), and an α/β domain (secondary pocket). The α/β domain (secondary pocket) has been shown to be important for the interaction with the CLOCK PAS B domain and maintains the BMAL1/CLOCK transcription factor in a repressed state to close the circadian feedback loop (
      • Michael A.K.
      • Fribourgh J.L.
      • Chelliah Y.
      • Sandate C.R.
      • Hura G.L.
      • Schneidman-Duhovny D.
      • Tripathi S.M.
      • Takahashi J.S.
      • Partch C.L.
      Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1.
      ).
      Although rare CRY1 variants have been previously shown to be associated with several disorders, the underlying mechanisms are yet to be characterized. Here, we not only investigated the potential functional impact of rare nonsynonymous SNPs on the function of CRY1 but also revealed the allosteric regulation between primary and secondary pockets of CRY1 by characterizing one of the rare mutants. The three variants, which map to important domains of the CRY1 and are predicted to affect its function, were selected from Ensembl and the 1000 Genomes project. Arg-348 and Gly-144 residues map to the surface; Arg-293 is located within the primary pocket, FAD-binding domain, and FBXL3 binding site of CRY1 (Fig. 1). We initially analyzed the effect of mutations on the t1/2 CRY1 protein using the CRY1::dLUC degradation assay. p.Arg348Cys and p.Gly144Val CRY1 variants had no effect on the stability of CRY1, whereas the p.Arg293His variant significantly increased the stability compared with WT CRY1 (Fig. 2A). Next, we utilized a cell-based rescue assay in Cry1−/−Cry2−/− MEF cells with a special Cry1 rescue plasmid to identify their effects on the circadian rhythm. Surprisingly, p.Arg293His CRY1 variant had longer t1/2 than the WT CRY1 and caused shorter period length with enhanced luminescence. This implies less repression activity (Fig. 2B), which is the indicator of weak binding of mutant CRY1 to BMAL1/CLOCK. Other two variants caused slight increase in the period length, which is consistent with previous findings indicating that mutation of residues on the surface of the α-helical domain have a tendency to change the repressor activity of mammalian CRYs (
      • McCarthy E.V.
      • Baggs J.E.
      • Geskes J.M.
      • Hogenesch J.B.
      • Green C.B.
      Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression.
      ). Despite earlier studies suggested that as the stability of CRY increases by either mutation or chemicals, period of circadian rhythm becomes longer; however, recent findings unveiled that not the stability but the ability of CRY to bind BMAL1/CLOCK is the primary factor in determining the period length (
      • Xu H.Y.
      • Gustafson C.L.
      • Sammons P.J.
      • Khan S.K.
      • Parsley N.C.
      • Ramanathan C.
      • Lee H.W.
      • Liu A.C.
      • Partch C.L.
      Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C terminus.
      ,
      • Rosensweig C.
      • Reynolds K.A.
      • Gao P.
      • Laothamatas I.
      • Shan Y.L.
      • Ranganathan R.
      • Takahashi J.S.
      • Green C.B.
      An evolutionary hotspot defines functional differences between CRYPTOCHROMES.
      ,
      • Patke A.
      • Murphy P.J.
      • Onat O.E.
      • Krieger A.C.
      • Özçelik T.
      • Campbell S.S.
      • Young M.W.
      Mutation of the human circadian clock gene CRY1 in familial delayed sleep phase disorder.
      ,
      • Hirano A.
      • Shi G.
      • Jones C.R.
      • Lipzen A.
      • Pennacchio L.A.
      • Xu Y.
      • Hallows W.C.
      • McMahon T.
      • Yamazaki M.
      • Ptáček L.J.
      • Fu Y.-H.
      A Cryptochrome 2 mutation yields advanced sleep phase in humans.
      ).
      To test binding of CRY to core clock proteins, pulldown experiment was performed with CRY1 and BMAL1/CLOCK in the presence and absence of PER2. As rescue assay results suggested, p.Arg293His CRY1 has deficiency to bind CLOCK, even in the presence of PER2 (Fig. 3). On the other hand, p.Gly144Val and p.Arg348C mutants showed comparable binding to core clock proteins with WT CRY1. Interestingly, functional characterization of p.Arg293His, which is located in the α12-helix and possibly interacts with the secondary pocket, revealed different properties than the other CRY1 variants. That is to say, t1/2 of the p.Arg293His CRY1 was longer than WT CRY1. Second, functional complementation with this variant in DKO-MEF resulted in a short circadian period phenotype with increased luminescence. Third, biochemical studies showed that this variant had reduced affinity to CLOCK. Interestingly, these features are more similar to CRY2 than CRY1 (
      • Rosensweig C.
      • Reynolds K.A.
      • Gao P.
      • Laothamatas I.
      • Shan Y.L.
      • Ranganathan R.
      • Takahashi J.S.
      • Green C.B.
      An evolutionary hotspot defines functional differences between CRYPTOCHROMES.
      ). Thus, we tested WT and p.Arg293His CRY1 and WT CRY2 in the same experimental set-up to determine the differences and similarities among these proteins. Although p.Arg293His CRY1 and WT CRY2 had comparable t1/2 (Fig. 4A), repression capacity, and binding to BMAL1/CLOCK in pulldown assays (Fig. 4, C and D), p.Arg293His caused a longer rhythm than WT CRY1 but shorter than WT CRY2 (Fig. 4B). Despite similarities between p.Arg293His CRY1 variant and WT CRY2 in various aspects, rescue assay exhibited that they have slight functionality differences in the circadian clock mechanism (Fig. 4B).
      To understand the dramatic change in the function of CRY1 upon Arg-293 to His-293 mutation and to assess the role of Arg-293 in CRY1, we employed molecular dynamics simulations. Computational analysis suggested that Arg-293 might be a critical residue for the regulation of the serine loop in CRY1, which is known to be important for CLOCK binding to the secondary pocket (
      • Fribourgh J.L.
      • Srivastava A.
      • Sandate C.R.
      • Michael A.K.
      • Hsu P.L.
      • Rakers C.
      • Nguyen L.T.
      • Torgrimson M.R.
      • Parico G.C.G.
      • Tripathi S.
      • Zheng N.
      • Lander G.C.
      • Hirota T.
      • Tama F.
      • Partch C.L.
      Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing.
      ). Replacing Arg-293 with His changes a highly dynamic serine loop into a less dynamic state and closes the gate of the secondary pocket (Fig. 5, BE), which may be responsible for the reduced binding of CLOCK to the mutant CRY1. Further analysis of our MD simulations of both variant and WT CRY1 in terms of the communication pathways suggested that mutation of Arg-293 to His changes the path to serine loop, which shows the allosteric regulation of secondary pocket availability by the primary pocket (Fig. 5, F and G).
      The rare variants characterized in this study were selected from published human SNPs, which are all in heterozygous state and, to our knowledge, have not yet been associated with any disease phenotype. It is possible that these variants may exhibit a disease phenotype in recessive state. Their effects can be further analyzed in mouse models by reverse phenotyping studies. In conclusion, we characterized the effect of rare human CRY1 SNPs on circadian clock function at the molecular level. Our findings suggest that Arg-293 is important for the allosteric regulation in CRY1 and has impact on the molecular clock.

      Experimental Procedures

      Site-directed mutagenesis

      For site-directed mutagenesis, Phusion polymerase–based quick-change method was used. Primers were designed using the recommended guidelines (
      • Doruk Y.U.
      • Yarparvar D.
      • Akyel Y.K.
      • Gul S.
      • Taskin A.C.
      • Yilmaz F.
      • Baris I.
      • Ozturk N.
      • Türkay M.
      • Ozturk N.
      • Okyar A.
      • Kavakli I.H.
      A CLOCK-binding small molecule disrupts the interaction between CLOCK and BMAL1 and enhances circadian rhythm amplitude.
      ). The PCR reaction mixtures contained 0.3 mm dNTP, 5 μl of 10× Phusion GC Buffer (Thermo Scientific), 3% DMSO, 1 μm each primer, 30 ng of the template plasmid (mCry1 in pcDNA4A), and 1 unit of Phusion DNA polymerase in a 50 μl of final volume. The reaction conditions were set to 98°C for 15 s, 66°C for 30 s, and 72°C for 4 min, 20 cycles. PCR products were visualized in 1% agarose gel. The samples with correct-sized bands were digested with 1 unit of FastDigest DpnI enzyme (Thermo Scientific) for 1 h at 37°C and then 5 μl of sample was transformed to Escherichia coli DH5α cells. Colonies were picked to culture and then plasmids were isolated via MiniPrep (Macherey Nagel). Sanger sequencing (Macrogen) was used to confirm the mutations.

      Cry-Luc construct generation

      To form Cry-Luc constructs, coding sequence of firefly luciferase in pG5luc plasmid (Addgene) was amplified with primers having NotI and XhoI flanking sites. Product size was verified by visualizing in agarose gel and product was isolated from the gel by using NucleoSpin PCR and Gel Purification Kit (Macherey Nagel). Purified fragments were cloned to pJET1.2/blunt vector (Thermo Scientific) using CloneJET PCR Cloning Kit (Thermo Scientific). 5 μl of sample was transformed to E. coli DH5α cells. Colonies were picked to culture and then plasmids were isolated via MiniPrep. Luc plasmid was double digested with NotI and XhoI; Cry pcDNA plasmids (WT or variant) were double digested with EcoRV and NotI FastDigest enzymes (Thermo Scientific). Cry plasmids were treated with FastAP (Thermo Scientific) to prevent self-annealing. After gel isolation the inserts and the destination vectors were cleaned and ligated by using T4 DNA ligase (Thermo Scientific). 5 μl of sample was transformed to E. coli DH5α cells. Colonies were picked to culture and then plasmids were isolated via MiniPrep. Presence of Cry and Luc were verified by double digestions with EcorV/NotI and NotI/XhoI, respectively.

      Repression assay of BMAL1/CLOCK-dependent transactivation

      Plasmids used in this study were gifted to us from Professor John Hogenesch from Cincinnati Children's Hospital. A mixture of 50 ng pSport6-Bmal1, 125 ng pSport6-CLOCK, 50 ng pGL3-mPer1::dLuc, 1 ng pRL-TK, 2.5 ng or lower doses of pcDNA4A-Cry1 (WT or variant), and empty pSport6 (to equalize the transfected amount of DNA) was reverse transfected to 4 × 104 HEK293T cells/well in a 96-well opaque plate via PEI transfection reagent. To determine the activity of BMAL1/CLOCK-dependent transactivation without a repressor, the mixture was supplemented with empty pcDNA4/myc-His A instead of Cry1 construct. In each independent experiment, transfections were triplicated for each condition. The plates were incubated for 24 h at 37°C, 5% CO2. Firefly luciferase and renilla luciferase expression was determined using the Dual-Glo Luciferase Assay System (Promega) using manufacturer's protocol.

      Pulldown assay

      4 × 105 HEK293T cells (per well) were seed to 6-well tissue plate 24 h before the transfection. Cells were transfected with Cry1-His-Myc (WT or variants) and Cry2-His-Myc in pcDNA4-A with FLAG-CMV-Bmal1, FLAG-CMV-CLOCK via PEI transfection reagent for IP with BMAL1, CLOCK, and CRY1 as described in Ref.
      • Doruk Y.U.
      • Yarparvar D.
      • Akyel Y.K.
      • Gul S.
      • Taskin A.C.
      • Yilmaz F.
      • Baris I.
      • Ozturk N.
      • Türkay M.
      • Ozturk N.
      • Okyar A.
      • Kavakli I.H.
      A CLOCK-binding small molecule disrupts the interaction between CLOCK and BMAL1 and enhances circadian rhythm amplitude.
      . FLAG-PER2-CMV plasmid was also transfected along with BMAL1, CLOCK, and Cry1 plasmids to pull down four clock proteins. For negative control BMAL1, CLOCK or BMAL1, CLOCK, and PER2 were transfected with empty pSport6 plasmid instead of Cry1. 24 h after transfection, cells were harvested via ice-cold PBS. After centrifugation pellets were lysed in 300 μl passive lysis buffer (PLB) (15 mm HEPES, 300 mm NaCl, 5 mm NaF, 1% Nonidet P-40 supplemented with fresh protease inhibitor) for 20 min on ice. To get rid of cell debris, samples were centrifuged for 15 min at 13,000 × g at +4°C. 10% of the supernatant was saved as input. 15 μl Ni-NTA Agarose resin (Qiagen) per sample was equilibrated by washing two times with TBS-300 (15 mm Tris, 300 mm NaCl) supplemented with 25 mm imidazole and two times with PLB. Remaining supernatant was added on to equilibrated resins with 25 mm imidazole. Cell lysates and resins were mixed for ∼2 h at 4°C to pull down CRYs. Resins were washed four times with TBS-300 (300 μl) having 35 mm imidazole. Proteins were isolated from resins by boiling in Laemmli Buffer (31.5 mm Tris-HCl, pH 6.8, buffer 10% glycerol, 1% SDS, 0.005% Bromphenol Blue, freshly added β-mercaptoethanol (5%)).
      Anti-FLAG antibody (Sigma A9469) was used to detect BMAL1, CLOCK, and PER2. Blots were stripped (Advansta Strip-It Buffer, R03722-D50) and incubated with anti-Myc antibody (Abcam, ab18185) and anti-CRY1 (Bethyl, catalog no. A302-614A) to detect CRYs as described in Ref.
      • Doruk Y.U.
      • Yarparvar D.
      • Akyel Y.K.
      • Gul S.
      • Taskin A.C.
      • Yilmaz F.
      • Baris I.
      • Ozturk N.
      • Türkay M.
      • Ozturk N.
      • Okyar A.
      • Kavakli I.H.
      A CLOCK-binding small molecule disrupts the interaction between CLOCK and BMAL1 and enhances circadian rhythm amplitude.
      . In all Western blot analyses, blots were incubated overnight with primary antibody. Mouse IgG κ binding protein (m-IgGκ BP) conjugated to horseradish peroxidase (HRP) (sc-516102) was used as the secondary antibody. Anti-beta Actin (Cell Signaling Technology, 8H10D10) was used to detect β-actin protein. To capture the chemiluminescent signals, WesternBright ECL HRP Substrate (Advansta K-12045-D20) and Bio-Rad ChemiDocTM Imaging System were used.

      CRY-LUC degradation assay

      40 ng of expression vector (Cry-Luc plasmid) was reverse transfected to 4 × 104 HEK293T cells on opaque 96-well plate with flat bottom via PEI transfection reagent. 48 h of post-transfection cells were treated with luciferin (0.4 mm final) and HEPES (10 mm final and pH 7.2). After 2 h, cycloheximide (20 μg/ml final) was added to wells to stop the protein synthesis. Plate was sealed with optically clear film and placed to Synergy H1. Luminescence readings were recorded every 10 min at 32°C for 24 h. t1/2 of protein was calculated via one-phase exponential decay fitting function in GraphPad Prism5 software.

      CRY degradation assay

      500 ng of WT CRY1 or p.Arg293His CRY1 plasmids in pcDNA4A-His-Myc vector were transfected to HEK293T cells on a 6-well plate with PEI transfection reagent. 24 h after transfection cells were treated with cycloheximide (100 μg/ml final) to stop protein synthesis. Cells were harvested at the time of cycloheximide treatment (0 h) after 4 and 8 h. Levels of proteins were analyzed via Western blotting by using anti-Myc or anti-β-actin to detect CRY and β-actin levels, respectively. Details of antibody treatments are as explained in the pulldown assay section above.

      Real-time bioluminescence rescue assay

      3 × 105 Cry1−/−/Cry2−/− mouse embryonic fibroblasts (DKO-MEFs) were seeded onto 35-mm clear tissue culture plates (cells were obtained from Prof. Ueda's group). Cells were transfected with 4000 ng pGL3-Per2-dLuc (luciferase reporter) and 150 ng of the Cry1 expression vector (pMU2-P(CRY1)-(intron 336), obtained from the Ueda group) (WT or variant), using FuGENE6 transfection reagent according to the manufacturer's protocols. After 72 h, cells were synchronized by treatment with 0.1 μm dexamethasone for 2 h. Then, the medium was replaced with bioluminescence recording medium (1% DMEM powder (w/v), 0.035% sodium bicarbonate, 0.35% D(+) glucose powder, 0.01 M HEPES buffer, 0.25% penicillin/streptomycin, 5% FBS), in which luciferin was freshly added (0.1 mm final concentration). Plates were sealed with vacuum grease and placed into a LumiCycle (Actimetrics). Bioluminescence was monitored for 70 s every 10 min for 5 days. Luminescence values were recorded and processed using the LumiCycle Analysis software. The first 20 h, data were discarded from the analysis because of high transient luminescence upon medium change. Period and amplitude were calculated for each sample. Each independent experiment was carried out with duplicate samples.

      Molecular dynamics simulations

      The protein structures of CRY1 and CRY2 were obtained via homology modeling. Complete PHR domain sequences of CRY1 and CRY2 were obtained from UniProt (mouse CRY1(P97784), mouse CRY2(Q9R194)). Sequences were submitted to the RaptorX web server to obtain protein structures for CRY1 and CRY2 (
      • Källberg M.
      • Wang H.P.
      • Wang S.
      • Peng J.
      • Wang Z.Y.
      • Lu H.
      • Xu J.B.
      Template-based protein structure modeling using the RaptorX web server.
      ). The structure of the CRY1 mutant R293H was also obtained by the same procedure. All structures were protonated with PROPKA using the PDB2PQR webserver (
      • Dolinsky T.J.
      • Nielsen J.E.
      • McCammon J.A.
      • Baker N.A.
      PDB2PQR: An automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations.
      ). The CHARMM36m force field was used for simulations (
      • Huang J.
      • Rauscher S.
      • Nawrocki G.
      • Ran T.
      • Feig M.
      • de Groot B.L.
      • Grubmüller H.
      • MacKerell Jr., A.D.
      CHARMM36m: an improved force field for folded and intrinsically disordered proteins.
      ). All three structures were solvated in a rectangular box using TIP3P water molecules and neutralized with the appropriate amount of sodium and chloride ions in visual molecular dynamics (VMD) (
      • Humphrey W.
      • Dalke A.
      • Schulten K.
      VMD: Visual molecular dynamics.
      ). Energy minimization was performed for 100,000 steps. Systems were gradually heated and equilibrated in NPT ensemble for 140 ps. Throughout equilibration, constraints on the proteins were gradually removed. After equilibration, a production run of 300 ns for WT, G144V, R293H, and R348C CRY1 was performed. Total of 1200 ns consisting of two independent production runs were performed for each protein. All production runs were performed at 310 K and under 1 atm pressure using a Langevin thermostat and a Langevin barostat, respectively. All simulations were performed using NAMD software on in-house, local machines (
      • Phillips J.C.
      • Braun R.
      • Wang W.
      • Gumbart J.
      • Tajkhorshid E.
      • Villa E.
      • Chipot C.
      • Skeel R.D.
      • Kalé L.
      • Schulten K.
      Scalable molecular dynamics with NAMD.
      ). To prepare trajectories for analysis, all trajectories were aligned to the initial structure, and the first 10 ns of all simulations were discarded. RMSD and RMSF calculations were performed using VMD. Each of the refined trajectories were saved as two multi-frame PDB files with 1 ns and 10-ps intervals for POVME and WISP, respectively, for further analysis. Calculation of the secondary pocket was performed using POVME 3.0 software (
      • Wagner J.R.
      • Sørensen J.
      • Hensley N.
      • Wong C.
      • Zhu C.
      • Perison T.
      • Amaro R.E.
      POVME 3.0: Software for mapping binding pocket flexibility.
      ). For all trajectories, the volume of the secondary pocket was calculated with a 1-ns interval. The Wilcoxon rank sum test was used to determine significance of the difference between the pocket volumes implemented in R (
      • Wickham H.
      ggplot2: Elegant Graphics for Data Analysis.
      ). Allosteric pathway analysis was performed using command line WISP 1.1 software (
      • Van Wart A.T.
      • Durrant J.
      • Votapka L.
      • Amaro R.E.
      Weighted Implementation of Suboptimal Paths (WISP): An optimized algorithm and tool for dynamical network analysis.
      ). Visualizations of pathways were performed using VMD software.

      Data Availability

      All data are contained within the manuscript.

      Acknowledgments

      We thank Emma Harris from the University of Edinburgh for critical reading of the manuscript. We are grateful to Prof. Hiroki Ueda and Andrew Liu for the generous gift of the Cry1−/−/Cry2−/− mouse embryonic fibroblasts and the Cry1 rescue vector. Finally, we thank Prof. Hogenesch for the generous gifts of clock-related plasmids and the support.

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