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The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock Jinkui Li a, b, Yongqi Huang a, b, Zhengding Su a, b, Sen Liu a, b, * a National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Industrial Fermentation (Ministry of Education), Hubei University of Technology, Wuhan, 430068, China b Institute of Biomedical and Pharmaceutical Sciences, Hubei Key Laboratory of Industrial Microbiology, Department of Biological and Food Engineering, Hubei University of Technology, Wuhan, 430068, China
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Article history: Received 16 December 2019 Accepted 13 January 2020 Available online xxx
Circadian rhythms are the endogenous oscillation of biological reactions and behaviors in most organisms on Earth. Circadian clocks are the pacemakers regulating circadian rhythms, and the transcriptiontranslation dependent feedback loop (TTFL) model was supposed to be the sole model of circadian clocks. However, recent years have witnessed rapid progresses in the study of non-TTFL circadian clocks. The cyanobacterial circadian clock consists of three proteins (KaiA, KaiB, and KaiC), and is extensively studied as a non-TTFL circadian clock model. Although containing only three proteins, the molecular mechanism of the KaiABC circadian clock remains elusive. We recently noticed that KaiA has an auto-inhibition conformation during the oscillation, but how this auto-inhibition is regulated is unclear. Here, we started from the design of light modulated KaiAs to investigate this mechanism. We designed different KaiA constructs fused with the light modulable LOV2 protein, and used light-modulated KaiAs to regulate the phosphorylation and dephosphorylation of KaiC. Our data indicated that the N-terminal domain of KaiA is important for KaiA’s reversible off/on switching during the unidirectional oscillation of the KaiABC system. This work provides an updated model to explain the molecular mechanism of the KaiABC circadian clock. © 2020 Elsevier Inc. All rights reserved.
Keywords: Circadian rhythm Post-translational circadian clock Protein design Oscillator LOV2
1. Introduction The circadian clock of cyanobacteria is the most thoroughly studied circadian clock model. One of the most attracting characteristics of the cyanobacterial circadian clock is this system can be re-constituted in vitro with purified clock proteins, i.e. KaiA, KaiB, and KaiC [1]. When these clock proteins are mixed with a specific ratio in a buffer solution containing ATP and Mg2þ, KaiC undergoes reversible phosphorylation and de-phosphorylation with a period of ~24 h [1]. The phosphorylation of KaiC depends on its autokinase activity, and KaiA binds with de-phosphorylated KaiC to stimulate its kinase activity [2]. The de-phosphorylation of KaiC is catalyzed by its phosphatase activity when KaiB binds to phosphorylated KaiC [2].
* Corresponding author. National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Industrial Fermentation (Ministry of Education), Hubei University of Technology, Wuhan, 430068, China. E-mail address: [email protected] (S. Liu).
Although the general molecular mechanism of the KaiABC system has been well known, the function of the N-terminal domain of KaiA (KaiA-ND) remains elusive. Previous studies demonstrated that KaiA has three functional domains (Fig. 1A): KaiA-ND is the amplitude amplifier, the central domain is the period adjuster, and the C-terminal domain (KaiA-CD, or KaiA180C) is the clock oscillator [3]. Structurally, KaiA forms a domain-swapped homodimer [4]. Two KaiA-CDs form two concaved interfaces for the binding of the C-terminal peptide of KaiC, and the dimerization of KaiA-CDs is independent of KaiA-ND and the central domain [5]. Recently, we demonstrated that the domain-swapped KaiA dimer has asymmetric dynamics, which provides a multi-step binding process for KaiC to sustain high binding specificity/affinity and robust clock oscillation [6]. Using KaiA from Synechococcus elongatus PCC 7942 (S. e.), We further demonstrated that the central domain can initiate the auto-inhibition of KaiA [7], which was in line with the results from KaiA of Thermosynechococcus elongatus (T. e.) [8]. We noticed that KaiA-CD alone sustains the hyper-phosphorylation status of KaiC, whereas KaiA135C (containing residues 135e284 for S. e.) undergoes auto-inhibition via its a5 helix, leading to the de-
https://doi.org/10.1016/j.bbrc.2020.01.072 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: J. Li et al., The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.072
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Fig. 1. The design strategy of the light-modulated KaiA. (A) The domain structures of the KaiA homodimer. Shown is the KaiA structure (PDB ID: 5C5E) of Synechococcus elongatus PCC 7942 (S. e.). The N-terminal domain contains the residues 1e134, the central domain contains the residues 135e179, and the C-terminal domain contains the residues 180e284. (B) The oscillated activity of KaiA. The “Active” and “KaiC-bound” structures were based on 5C5E (PDB ID). The “Auto-inhibited” structure was based on 5JWR (PDB ID). The structural elements of KaiA are colored as in (A). The C-terminal tail of KaiC is colored in cyan. (C) Sequence comparison of the a5 helix of KaiA from S. e. and the Ja-helix of LOV2 (Avena sativa), including the a5 helix of KaiA from T. e.. The three colored residues are selected for mutagenesis. The four residues marked by gray background are the important residues for KaiA forming complexes with KaiB and KaiC [8]. (D) The location of the three designed residues in the a5 helix of KaiA (PDB ID: 5JWR). The three key residues are shown in sticks and dots. (E) The location of the three designed residues in the Ja helix of LOV2 (PDB ID: 2V0U). The three mutation residues are shown in sticks and dots. (F) The design strategy in this work. Upon light excitation, the mutated Ja-helix (Ja*) dissociates from the core domain of LOV2, and then binds to the concaved pocket of the KaiA homodimer and inhibits KaiA’s activity. This process is reversed when without light. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
phosphorylation of KaiC [7]. Although with these proceedings, how KaiA-ND performs its function during the oscillation is not clear yet. Additionally, the function of KaiB is worth more investigations. Previous studies demonstrated that KaiB antagonizes KaiA’s function, but KaiB’s binding with the phosphorylated KaiC does not change KaiC’s phosphatase activity [9]. KaiB was firstly proposed to bind with the CII domain of KaiC, but later studies discussed that the main binding site of KaiB on KaiC is the CI domain [10]. More recently, the structures of the KaiA/KaiB/KaiC complex confirmed that KaiB binds to the CI domain of KaiC [8,11]. For antagonizing KaiA, a fold-switch change in the structure of KaiB is important [12]. In spite of these progresses, it is still elusive regarding if KaiB has any other unknown roles. Based on previous studies, KaiA-ND is a pseudo-receiver and KaiB does not directly regulate the dephosphorylation of KaiC [13]. Therefore, we intended to redesign KaiA to elucidate the regulation mechanism of KaiA-ND. We hoped to substitute KaiA-ND with a real light receiver to switch KaiA’s activity without KaiB. Through protein design, we obtained light modulated KaiA constructs. Using these constructs, we showed that withouth KaiB and KaiA-ND, KaiA and KaiC could not form phosphorylation/de-phosphorylation oscillations. Based on these data, we proposed a potential model to deepen our understanding on the functions of KaiA-ND and KaiB in the KaiABC circadian oscillator.
synthesized by Biotech Co., Ltd. (Shanghai, China). The DNA sequence of the oat (avena sativa) LOV2 was cloned from pTriExmCherry-PA-Rac1 (Addgene). The pGEX-6P-1 plasmids containing the coding sequences of the wild-type KaiA, KaiB and KaiC proteins of S.e. PCC 7942 were kindly provided by Dr. Carl Johnson (Vanderbilt University). The primers LOV2-F and LOV2-R/LOV2-T535LR/LOV2-T535L-I532S-R/LOV2-T535L-I532S-G528Q-R were used to clone and mutate LOV2 with a stop codon through polymerase chain reaction (PCR). These constructs were used for express LOV2 proteins. To prepare the constructs for expression LOV2-fused KaiA proteins, the primers LOV2-F and LOV2-ins-R/LOV2-T535L-ins-R/ LOV2-T535L-I532S-ins-R/LOV2-T535L-I532S-G528Q-ins-R were used. These constructs had no stop codons after the coding sequence of LOV2 and were inserted in pET28a using the Nde I site and the BamH I site. Then, the DNA sequences of KaiA180C, KaiA135C, and KaiA166C were digested with BamH I and Not I from the previous pGEX-6P-1-KaiA180C, pGEX-6P-1-KaiA135C, and pGEX-6P-1-KaiA166C plasmids [7] respectively. These KaiA coding sequences contained stop codons, so these KaiA coding sequences were inserted into the pET28a plasmids containing the LOV2 coding sequences without stop codons after the digestion of BamH I and Not I to obtain the plasmids for expression LOV2-fused KaiA proteins. All plasmids and mutations were verified with DNA sequencing.
2. Materials and methods 2.2. Protein expression and purification 2.1. Plasmid construction and mutagenesis The primers used for DNA cloning are shown in Table S1 and
The coding sequences of the wild-type KaiA, KaiB, and KaiC were inserted in pGEX-6P-1, so these proteins were expressed as
Please cite this article as: J. Li et al., The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.072
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GST-tagged products in E. coli BL21(DE3) as previously described [6,14]. GST-tagged proteins were purified using GST affinity beads, and then the GST tag was removed with the PreScission protease. The tag-free proteins were further purified with a size exclusion column. For the expression of the pET28a based constructs, the plasmids were transferred into E. coli BL21(DE3). When cultured in LB (LuriaBertani) liquid medium until the OD value reached 0.6e0.8, IPTG (Isopropyl-b-D-thiogalactoside) was added to a final concentration of 100 mM/L to induce protein expression for 16 h at 16 C overnight. These proteins were expressed with 6xHis tags. The proteins were purified by Ni-NTA affinity chromatography, followed by a further purification using a size exclusion column. 2.3. Regulation of the phosphorylation of KaiC The concentrations of the purified proteins were determined by the Bradford assay. To analyze the phosphorylation state of KaiC, the proteins were incubated in the reconstitution buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM ATP, 5 mM MgCl2 and 0.01% Tween-20) alone or with indicated proteins. For the wild-type Kai proteins, the molar ratio of KaiA:KaiB:KaiC was 1:1:2. Using this as a reference, the ratio of KaiA:KaiC was 1:2 when any KaiA protein or LOV2 was co-incubated with KaiC. The solutions were incubated at 30 C, and samples were collected at indicated time. 2.4. Light excitation analysis The light excitation and absorption experiments of LOV2-Ja were carried out in a blue light LED source with the wavelength of 460 nm. The proteins were 3 mM in the reconstitution buffer. After light excitation for indicated time at 30 C, the light absorbance changes of the samples were measured at 450 nm on a fluorescence microplate reader (BioTeK) at 30 C. The reading was performed every 10 s and last for 5 min. The absorbance change was calculated as the ratio of the light-excited sample and the dark sample.
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and T535L. In the auto-inhibited KaiA, these residues faces the binding site, so they are supposed to provide key interactions. With these mutations, we hoped that the Ja helix binds with the core domain of LOV2 in darkness but binds to KaiA under light to form auto-inhibited KaiA and inhibit KaiA-KaiC interaction (Fig. 1F). 3.2. The identification of light modulated KaiA Combining the identified mutation sites, we designed three LOV2 proteins with Ja mutations (Fig. 2A): LOV2-Ja1: T535L; LOV2Ja2: I532S-T535L; LOV2-Ja3: G528Q-I532S-T535L. Then, based on our previous study, we used three engineered KaiA (S. e.) constructs to make the fused proteins (Fig. 2A): KaiA180C: containing the residues 180e284; KaiA166C: containing the residues 166e284; KaiA135C: containing the residues 135e284. Because KaiA135C contains the a5 helix, we only fused it with the wild-type LOV2 protein, hoping that the conformational change of LOV2 could cause space hindrance to release the auto-inhibited conformation. With these thirteen protein constructs, we first tested if the LOV2 domains are still functional. The absorbance of LOV2 at 450 nm will decrease when stimulated by light (460 nm), and the absorbance will quickly recover when the light disappears [18]. As shown in Fig. 2B, although the mutation in the Ja helix caused varied responses of LOV2, the mutants and the KaiA-fused constructs still showed this response. As expected, the fusion of KaiA135C did not significantly disrupt the function of LOV2. Then we tested if these constructs still keep the function of KaiA in stimulating KaiC phosphorylation. As shown in Fig. 2C, the constructs of KaiA180C and KaiA166C stimulated KaiC phosphorylation and sustained its hyper-phosphorylation state, but the KaiA135C construct could not sustain the high phosphorylation state of KaiC after it stimulated KaiC phosphorylation. Next, we continued to test if light would induce KaiA’s auto-inhibition and cause or accelerate KaiC’s de-phosphorylation. As shown in Fig. 2D, among all constructs, KaiC had faster de-phosphorylation only with LOV2-Ja3KaiA180C upon light excitation. This result indicated that LOV2-Ja3KaiA180C could be modulated by light.
2.5. SDS-PAGE gel analysis and quantitative calculation 3.3. The functional exploration of LM-KaiA The collected samples were analyzed with 8% SDS-PAGE gels. The SDS-PAGE gels were analyzed using Image J (NIH, USA) as previously described [15]Li:2019ik}. The percentage of phosphorylated KaiC was calculated and the data was calculated in each lane. 3. Results 3.1. The design strategy of light modulated KaiA (LM-KaiA) The recent studies from others [8,11] and our group [7] suggested that KaiA undergoes oscillated conformational changes that regulate its active-inactive states (Fig. 1B). The C-terminal tail of KaiC binds to the concaved site of two KaiA-CDs with an a-helix conformation [16]. The same site is used for the auto-inhibition of KaiA by its central domain (containing the a5 helix) through forming a competitive a-helix conformation [8]. As a widely used optogenetic tool, the LOV2 protein has a Ja helix that undergoes a reversible bound-unbound state change upon repeated light/dark process [17]. Based on our recent functional studies of KaiA, we chose to take advantage of the LOV2 protein to design light modulated KaiAs (LM-KaiAs). The sequence comparison of the a5 helix of KaiA and the Jahelix of LOV2 showed that these two helices have very similar residue compositions (Fig. 1C). Looking into the structure of the auto-inhibited KaiA (Fig. 1D) and the LOV2 protein (Fig. 1E), we chose three residues in the Ja helix for mutagenesis: G528Q, I532S,
To test if LOV2-Ja3-KaiA180C would induce the reversible phosphorylation/de-phosphorylation of KaiC, we co-incubated this protein with KaiC in 8-h darkness/light cycles (Fig. 3A). After phosphorylation and then de-phosphorylation, KaiC failed to rephosphorylate again. To exclude the possibility that lowphosphorylation KaiC lost activity after long-time incubation, we incubated KaiC alone for 12 h to have it de-phosphorylated, and then added KaiA to the solution. As shown in Fig. 3B, both the wildtype KaiA and LOV2-Ja3-KaiA180C stimulated the phosphorylation of KaiC. However, when KaiC was firstly co-incubated with LOV2Ja3-KaiA180C, neither the wild-type KaiA or LOV2-Ja3-KaiA180C could re-stimulate the phosphorylation after KaiC dephosphorylated (Fig. 3C). Furthermore, our test showed that adding KaiB to the co-incubation system of LOV2-Ja3-KaiA180C and KaiC did not help the re-phosphorylation of KaiC as well (Fig. 3D). 4. Discussion Circadian rhythms are the intrinsic rhythms of most organisms on Earth. The acquirement of circadian rhythms endows organisms with evolutionary advantages [19]. In human, circadian rhythms are closely related with health, aging, and cancers [20]. Recent years have witnessed increased attention on the study of circadian rhythms since the Nobel Prize in Medicine was awarded to Jeffrey C. Hall, Michael Rosbash and Michael W. Young for their work on the
Please cite this article as: J. Li et al., The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.072
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Fig. 2. The experimental identification of the light modulated KaiAs. (A) The protein constructs tested in this work. The different structural elements are colored as in Fig. 1. The mutated sites in the Ja helix are represented by colored bars. The residue numbers of KaiA are shown at the bottom. (B) The test of the light absorbance recovery (450 nm) of the LOV2 protein in different constructs after light stimulation. (C) The test of the function of these constructs in stimulating KaiC’s phosphorylation in darkness. (D) The test of the function of these constructs in light-stimulated functional changes. The SDS-PAGE gels are shown in Fig. S1. In all pictures, the protein bands in the SDS-PAGE gels are KaiC, with the upper band identified as the phosphorylated KaiC, and the bottom band identified as the de-phosphorylated KaiC. The gels were quantitatively processed in Image J and the percentage of the phosphorylated KaiC were shown in the corresponding plots.
molecular regulation of circadian rhythms in multicellular organisms in 2017 [21]. In multicellular organisms, circadian rhythms are controlled by circadian clocks, which generally consist of multilevel feedback regulations including DNA transcription, RNA translation, and protein modification [19]. This transcription-translation feedback loop (TTFL) model has been extensively studied since it was supposed to be the sole mechanism of circadian clocks in most organisms including human [22]. However, recent studies revealed that there exist conserved non-TTFL regulations of the circadian clocks of all domains of life [23]. It was then demonstrated that the coupling of TTFL and non-TTFL regulations is a key feature of robust circadian rhythms [24,25]Zwicker:2010fe}. Therefore, it is of great importance to study the molecular mechanism of non-TTFL circadian clocks as well. The circadian clock of cyanobacteria is the first verified circadian clock independent of TTFLs [26]. The cyanobacterial circadian clock contains only three proteins (KaiA, KaiB, and KaiC), but its mechanism is far more complicated than its composition. In this work, we adopted a protein design strategy to study the functional switch
of KaiA during this process. We wanted to design a KaiA protein that could be reversibly switched off and on with light, but unfortunately, we did not fully achieve this goal in this work. Our design strategy gave us light regulated KaiA constructs, but the constructs did not work well in the in vitro reconstitution of the KaiABC system as expected. However, our data indicated that the N-terminal domain of KaiA is an indispensable player in the function switching of KaiA. Most interestingly, our data indicated that when KaiC and LOV2-Ja3-KaiA180C were co-incubated, KaiC was locked in a state prohibiting its re-phosphorylation. Furthermore, this locked state could not be rescued by the wild-type KaiA or KaiB. Combining our data, a likely explanation is once LOV2-Ja3KaiA180C became auto-inhibited, it did not fully release from KaiC; meanwhile, our previous study demonstrated that conformational changes provide stronger KaiA-KaiC interaction [6], so fresh KaiA could not bind with hypo-phosphorylated KaiC and stimulate its phosphorylation. With these data and recent studies from others [8,11] and our group [7], we proposed a possible model (Fig. 3E) of the KaiABC system, in which KaiB’s function is not limited to
Please cite this article as: J. Li et al., The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.072
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Fig. 3. The functional exploration of LOV2-Ja3-KaiA180C. (A) LOV2-Ja3-KaiA180C and KaiC were co-incubated in cycled dark/light environments to test if KaiC would have oscillated phosphorylation/de-phosphorylation changes. The green box indicates the time window for constant light. (B) KaiC was incubated at 30 C for 12 h to de-phosphorylate, then KaiA proteins (the wild-type KaiA, or LOV2-Ja3-KaiA180C) were added to stimulate KaiC’s phosphorylation. (C) LOV2-Ja3-KaiA180C and KaiC were co-incubated for 24 h, during which KaiC phosphorylated and then de-phosphorylated. At the indicated time point (24 h), additional KaiA proteins (the wild-type KaiA, or LOV2-Ja3-KaiA180C) were added to the solution to test if the fresh KaiA proteins would stimulate the phosphorylation of KaiC. (D) LOV2-Ja3-KaiA180C, KaiB and KaiC were co-incubated to test if KaiB could induce the rephosphorylation of KaiC. (E) A working model of the co-operation of KaiA and KaiB during the clock oscillation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
antagonize KaiA and induce the de-phosphorylation of KaiC, but it also chaperones the auto-inhibited KaiA and helps restore KaiA’s active conformation. During this process, KaiB is like a “hand” whereas the N-terminal domain of KaiA is a “latch”, so that KaiB flips the handle to turn off and turn on KaiA’s function sequentially (unidirectionally), which guarantees the unidirectional running of the clock in addition to the sequential phosphorylation/dephosphorylation of KaiC [27]. Recent studies showed that non-TTFL circadian clocks universally exist in many organisms, and the role of non-TTFL circadian clocks is indispensable for robust circadian rhythms [24,25]. Besides being an important model for non-TTFL circadian clocks, the KaiABC circadian clock is also intriguing for studying biological oscillators and protein-protein interaction [28]. We hope our work will help elucidating the function and mechanism of the KaiABC circadian clock. Declaration of competing interest The authors declare no competing financial interests. Acknowledgements We would like to thank Dr. Carl Johnson (Vanderbilt University) for generously providing the plasmids containing the Kai proteins. S. L. was supported by the grants from National Natural Science Foundation of China (31670768, 31971150), Hubei Provincial Science and Technology Department (2019CFA069), Wuhan Science and Technology Bureau of China (2018060401011319), and the
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The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock Jinkui Li a, b, Yongqi Huang a, b, Zhengding Su a, b, Sen Liu a, b, * a National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Industrial Fermentation (Ministry of Education), Hubei University of Technology, Wuhan, 430068, China b Institute of Biomedical and Pharmaceutical Sciences, Hubei Key Laboratory of Industrial Microbiology, Department of Biological and Food Engineering, Hubei University of Technology, Wuhan, 430068, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2019 Accepted 13 January 2020 Available online xxx
Circadian rhythms are the endogenous oscillation of biological reactions and behaviors in most organisms on Earth. Circadian clocks are the pacemakers regulating circadian rhythms, and the transcriptiontranslation dependent feedback loop (TTFL) model was supposed to be the sole model of circadian clocks. However, recent years have witnessed rapid progresses in the study of non-TTFL circadian clocks. The cyanobacterial circadian clock consists of three proteins (KaiA, KaiB, and KaiC), and is extensively studied as a non-TTFL circadian clock model. Although containing only three proteins, the molecular mechanism of the KaiABC circadian clock remains elusive. We recently noticed that KaiA has an auto-inhibition conformation during the oscillation, but how this auto-inhibition is regulated is unclear. Here, we started from the design of light modulated KaiAs to investigate this mechanism. We designed different KaiA constructs fused with the light modulable LOV2 protein, and used light-modulated KaiAs to regulate the phosphorylation and dephosphorylation of KaiC. Our data indicated that the N-terminal domain of KaiA is important for KaiA’s reversible off/on switching during the unidirectional oscillation of the KaiABC system. This work provides an updated model to explain the molecular mechanism of the KaiABC circadian clock. © 2020 Elsevier Inc. All rights reserved.
Keywords: Circadian rhythm Post-translational circadian clock Protein design Oscillator LOV2
1. Introduction The circadian clock of cyanobacteria is the most thoroughly studied circadian clock model. One of the most attracting characteristics of the cyanobacterial circadian clock is this system can be re-constituted in vitro with purified clock proteins, i.e. KaiA, KaiB, and KaiC [1]. When these clock proteins are mixed with a specific ratio in a buffer solution containing ATP and Mg2þ, KaiC undergoes reversible phosphorylation and de-phosphorylation with a period of ~24 h [1]. The phosphorylation of KaiC depends on its autokinase activity, and KaiA binds with de-phosphorylated KaiC to stimulate its kinase activity [2]. The de-phosphorylation of KaiC is catalyzed by its phosphatase activity when KaiB binds to phosphorylated KaiC [2].
* Corresponding author. National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Industrial Fermentation (Ministry of Education), Hubei University of Technology, Wuhan, 430068, China. E-mail address: [email protected] (S. Liu).
Although the general molecular mechanism of the KaiABC system has been well known, the function of the N-terminal domain of KaiA (KaiA-ND) remains elusive. Previous studies demonstrated that KaiA has three functional domains (Fig. 1A): KaiA-ND is the amplitude amplifier, the central domain is the period adjuster, and the C-terminal domain (KaiA-CD, or KaiA180C) is the clock oscillator [3]. Structurally, KaiA forms a domain-swapped homodimer [4]. Two KaiA-CDs form two concaved interfaces for the binding of the C-terminal peptide of KaiC, and the dimerization of KaiA-CDs is independent of KaiA-ND and the central domain [5]. Recently, we demonstrated that the domain-swapped KaiA dimer has asymmetric dynamics, which provides a multi-step binding process for KaiC to sustain high binding specificity/affinity and robust clock oscillation [6]. Using KaiA from Synechococcus elongatus PCC 7942 (S. e.), We further demonstrated that the central domain can initiate the auto-inhibition of KaiA [7], which was in line with the results from KaiA of Thermosynechococcus elongatus (T. e.) [8]. We noticed that KaiA-CD alone sustains the hyper-phosphorylation status of KaiC, whereas KaiA135C (containing residues 135e284 for S. e.) undergoes auto-inhibition via its a5 helix, leading to the de-
https://doi.org/10.1016/j.bbrc.2020.01.072 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: J. Li et al., The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.072
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Fig. 1. The design strategy of the light-modulated KaiA. (A) The domain structures of the KaiA homodimer. Shown is the KaiA structure (PDB ID: 5C5E) of Synechococcus elongatus PCC 7942 (S. e.). The N-terminal domain contains the residues 1e134, the central domain contains the residues 135e179, and the C-terminal domain contains the residues 180e284. (B) The oscillated activity of KaiA. The “Active” and “KaiC-bound” structures were based on 5C5E (PDB ID). The “Auto-inhibited” structure was based on 5JWR (PDB ID). The structural elements of KaiA are colored as in (A). The C-terminal tail of KaiC is colored in cyan. (C) Sequence comparison of the a5 helix of KaiA from S. e. and the Ja-helix of LOV2 (Avena sativa), including the a5 helix of KaiA from T. e.. The three colored residues are selected for mutagenesis. The four residues marked by gray background are the important residues for KaiA forming complexes with KaiB and KaiC [8]. (D) The location of the three designed residues in the a5 helix of KaiA (PDB ID: 5JWR). The three key residues are shown in sticks and dots. (E) The location of the three designed residues in the Ja helix of LOV2 (PDB ID: 2V0U). The three mutation residues are shown in sticks and dots. (F) The design strategy in this work. Upon light excitation, the mutated Ja-helix (Ja*) dissociates from the core domain of LOV2, and then binds to the concaved pocket of the KaiA homodimer and inhibits KaiA’s activity. This process is reversed when without light. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
phosphorylation of KaiC [7]. Although with these proceedings, how KaiA-ND performs its function during the oscillation is not clear yet. Additionally, the function of KaiB is worth more investigations. Previous studies demonstrated that KaiB antagonizes KaiA’s function, but KaiB’s binding with the phosphorylated KaiC does not change KaiC’s phosphatase activity [9]. KaiB was firstly proposed to bind with the CII domain of KaiC, but later studies discussed that the main binding site of KaiB on KaiC is the CI domain [10]. More recently, the structures of the KaiA/KaiB/KaiC complex confirmed that KaiB binds to the CI domain of KaiC [8,11]. For antagonizing KaiA, a fold-switch change in the structure of KaiB is important [12]. In spite of these progresses, it is still elusive regarding if KaiB has any other unknown roles. Based on previous studies, KaiA-ND is a pseudo-receiver and KaiB does not directly regulate the dephosphorylation of KaiC [13]. Therefore, we intended to redesign KaiA to elucidate the regulation mechanism of KaiA-ND. We hoped to substitute KaiA-ND with a real light receiver to switch KaiA’s activity without KaiB. Through protein design, we obtained light modulated KaiA constructs. Using these constructs, we showed that withouth KaiB and KaiA-ND, KaiA and KaiC could not form phosphorylation/de-phosphorylation oscillations. Based on these data, we proposed a potential model to deepen our understanding on the functions of KaiA-ND and KaiB in the KaiABC circadian oscillator.
synthesized by Biotech Co., Ltd. (Shanghai, China). The DNA sequence of the oat (avena sativa) LOV2 was cloned from pTriExmCherry-PA-Rac1 (Addgene). The pGEX-6P-1 plasmids containing the coding sequences of the wild-type KaiA, KaiB and KaiC proteins of S.e. PCC 7942 were kindly provided by Dr. Carl Johnson (Vanderbilt University). The primers LOV2-F and LOV2-R/LOV2-T535LR/LOV2-T535L-I532S-R/LOV2-T535L-I532S-G528Q-R were used to clone and mutate LOV2 with a stop codon through polymerase chain reaction (PCR). These constructs were used for express LOV2 proteins. To prepare the constructs for expression LOV2-fused KaiA proteins, the primers LOV2-F and LOV2-ins-R/LOV2-T535L-ins-R/ LOV2-T535L-I532S-ins-R/LOV2-T535L-I532S-G528Q-ins-R were used. These constructs had no stop codons after the coding sequence of LOV2 and were inserted in pET28a using the Nde I site and the BamH I site. Then, the DNA sequences of KaiA180C, KaiA135C, and KaiA166C were digested with BamH I and Not I from the previous pGEX-6P-1-KaiA180C, pGEX-6P-1-KaiA135C, and pGEX-6P-1-KaiA166C plasmids [7] respectively. These KaiA coding sequences contained stop codons, so these KaiA coding sequences were inserted into the pET28a plasmids containing the LOV2 coding sequences without stop codons after the digestion of BamH I and Not I to obtain the plasmids for expression LOV2-fused KaiA proteins. All plasmids and mutations were verified with DNA sequencing.
2. Materials and methods 2.2. Protein expression and purification 2.1. Plasmid construction and mutagenesis The primers used for DNA cloning are shown in Table S1 and
The coding sequences of the wild-type KaiA, KaiB, and KaiC were inserted in pGEX-6P-1, so these proteins were expressed as
Please cite this article as: J. Li et al., The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.072
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GST-tagged products in E. coli BL21(DE3) as previously described [6,14]. GST-tagged proteins were purified using GST affinity beads, and then the GST tag was removed with the PreScission protease. The tag-free proteins were further purified with a size exclusion column. For the expression of the pET28a based constructs, the plasmids were transferred into E. coli BL21(DE3). When cultured in LB (LuriaBertani) liquid medium until the OD value reached 0.6e0.8, IPTG (Isopropyl-b-D-thiogalactoside) was added to a final concentration of 100 mM/L to induce protein expression for 16 h at 16 C overnight. These proteins were expressed with 6xHis tags. The proteins were purified by Ni-NTA affinity chromatography, followed by a further purification using a size exclusion column. 2.3. Regulation of the phosphorylation of KaiC The concentrations of the purified proteins were determined by the Bradford assay. To analyze the phosphorylation state of KaiC, the proteins were incubated in the reconstitution buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM ATP, 5 mM MgCl2 and 0.01% Tween-20) alone or with indicated proteins. For the wild-type Kai proteins, the molar ratio of KaiA:KaiB:KaiC was 1:1:2. Using this as a reference, the ratio of KaiA:KaiC was 1:2 when any KaiA protein or LOV2 was co-incubated with KaiC. The solutions were incubated at 30 C, and samples were collected at indicated time. 2.4. Light excitation analysis The light excitation and absorption experiments of LOV2-Ja were carried out in a blue light LED source with the wavelength of 460 nm. The proteins were 3 mM in the reconstitution buffer. After light excitation for indicated time at 30 C, the light absorbance changes of the samples were measured at 450 nm on a fluorescence microplate reader (BioTeK) at 30 C. The reading was performed every 10 s and last for 5 min. The absorbance change was calculated as the ratio of the light-excited sample and the dark sample.
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and T535L. In the auto-inhibited KaiA, these residues faces the binding site, so they are supposed to provide key interactions. With these mutations, we hoped that the Ja helix binds with the core domain of LOV2 in darkness but binds to KaiA under light to form auto-inhibited KaiA and inhibit KaiA-KaiC interaction (Fig. 1F). 3.2. The identification of light modulated KaiA Combining the identified mutation sites, we designed three LOV2 proteins with Ja mutations (Fig. 2A): LOV2-Ja1: T535L; LOV2Ja2: I532S-T535L; LOV2-Ja3: G528Q-I532S-T535L. Then, based on our previous study, we used three engineered KaiA (S. e.) constructs to make the fused proteins (Fig. 2A): KaiA180C: containing the residues 180e284; KaiA166C: containing the residues 166e284; KaiA135C: containing the residues 135e284. Because KaiA135C contains the a5 helix, we only fused it with the wild-type LOV2 protein, hoping that the conformational change of LOV2 could cause space hindrance to release the auto-inhibited conformation. With these thirteen protein constructs, we first tested if the LOV2 domains are still functional. The absorbance of LOV2 at 450 nm will decrease when stimulated by light (460 nm), and the absorbance will quickly recover when the light disappears [18]. As shown in Fig. 2B, although the mutation in the Ja helix caused varied responses of LOV2, the mutants and the KaiA-fused constructs still showed this response. As expected, the fusion of KaiA135C did not significantly disrupt the function of LOV2. Then we tested if these constructs still keep the function of KaiA in stimulating KaiC phosphorylation. As shown in Fig. 2C, the constructs of KaiA180C and KaiA166C stimulated KaiC phosphorylation and sustained its hyper-phosphorylation state, but the KaiA135C construct could not sustain the high phosphorylation state of KaiC after it stimulated KaiC phosphorylation. Next, we continued to test if light would induce KaiA’s auto-inhibition and cause or accelerate KaiC’s de-phosphorylation. As shown in Fig. 2D, among all constructs, KaiC had faster de-phosphorylation only with LOV2-Ja3KaiA180C upon light excitation. This result indicated that LOV2-Ja3KaiA180C could be modulated by light.
2.5. SDS-PAGE gel analysis and quantitative calculation 3.3. The functional exploration of LM-KaiA The collected samples were analyzed with 8% SDS-PAGE gels. The SDS-PAGE gels were analyzed using Image J (NIH, USA) as previously described [15]Li:2019ik}. The percentage of phosphorylated KaiC was calculated and the data was calculated in each lane. 3. Results 3.1. The design strategy of light modulated KaiA (LM-KaiA) The recent studies from others [8,11] and our group [7] suggested that KaiA undergoes oscillated conformational changes that regulate its active-inactive states (Fig. 1B). The C-terminal tail of KaiC binds to the concaved site of two KaiA-CDs with an a-helix conformation [16]. The same site is used for the auto-inhibition of KaiA by its central domain (containing the a5 helix) through forming a competitive a-helix conformation [8]. As a widely used optogenetic tool, the LOV2 protein has a Ja helix that undergoes a reversible bound-unbound state change upon repeated light/dark process [17]. Based on our recent functional studies of KaiA, we chose to take advantage of the LOV2 protein to design light modulated KaiAs (LM-KaiAs). The sequence comparison of the a5 helix of KaiA and the Jahelix of LOV2 showed that these two helices have very similar residue compositions (Fig. 1C). Looking into the structure of the auto-inhibited KaiA (Fig. 1D) and the LOV2 protein (Fig. 1E), we chose three residues in the Ja helix for mutagenesis: G528Q, I532S,
To test if LOV2-Ja3-KaiA180C would induce the reversible phosphorylation/de-phosphorylation of KaiC, we co-incubated this protein with KaiC in 8-h darkness/light cycles (Fig. 3A). After phosphorylation and then de-phosphorylation, KaiC failed to rephosphorylate again. To exclude the possibility that lowphosphorylation KaiC lost activity after long-time incubation, we incubated KaiC alone for 12 h to have it de-phosphorylated, and then added KaiA to the solution. As shown in Fig. 3B, both the wildtype KaiA and LOV2-Ja3-KaiA180C stimulated the phosphorylation of KaiC. However, when KaiC was firstly co-incubated with LOV2Ja3-KaiA180C, neither the wild-type KaiA or LOV2-Ja3-KaiA180C could re-stimulate the phosphorylation after KaiC dephosphorylated (Fig. 3C). Furthermore, our test showed that adding KaiB to the co-incubation system of LOV2-Ja3-KaiA180C and KaiC did not help the re-phosphorylation of KaiC as well (Fig. 3D). 4. Discussion Circadian rhythms are the intrinsic rhythms of most organisms on Earth. The acquirement of circadian rhythms endows organisms with evolutionary advantages [19]. In human, circadian rhythms are closely related with health, aging, and cancers [20]. Recent years have witnessed increased attention on the study of circadian rhythms since the Nobel Prize in Medicine was awarded to Jeffrey C. Hall, Michael Rosbash and Michael W. Young for their work on the
Please cite this article as: J. Li et al., The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.072
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Fig. 2. The experimental identification of the light modulated KaiAs. (A) The protein constructs tested in this work. The different structural elements are colored as in Fig. 1. The mutated sites in the Ja helix are represented by colored bars. The residue numbers of KaiA are shown at the bottom. (B) The test of the light absorbance recovery (450 nm) of the LOV2 protein in different constructs after light stimulation. (C) The test of the function of these constructs in stimulating KaiC’s phosphorylation in darkness. (D) The test of the function of these constructs in light-stimulated functional changes. The SDS-PAGE gels are shown in Fig. S1. In all pictures, the protein bands in the SDS-PAGE gels are KaiC, with the upper band identified as the phosphorylated KaiC, and the bottom band identified as the de-phosphorylated KaiC. The gels were quantitatively processed in Image J and the percentage of the phosphorylated KaiC were shown in the corresponding plots.
molecular regulation of circadian rhythms in multicellular organisms in 2017 [21]. In multicellular organisms, circadian rhythms are controlled by circadian clocks, which generally consist of multilevel feedback regulations including DNA transcription, RNA translation, and protein modification [19]. This transcription-translation feedback loop (TTFL) model has been extensively studied since it was supposed to be the sole mechanism of circadian clocks in most organisms including human [22]. However, recent studies revealed that there exist conserved non-TTFL regulations of the circadian clocks of all domains of life [23]. It was then demonstrated that the coupling of TTFL and non-TTFL regulations is a key feature of robust circadian rhythms [24,25]Zwicker:2010fe}. Therefore, it is of great importance to study the molecular mechanism of non-TTFL circadian clocks as well. The circadian clock of cyanobacteria is the first verified circadian clock independent of TTFLs [26]. The cyanobacterial circadian clock contains only three proteins (KaiA, KaiB, and KaiC), but its mechanism is far more complicated than its composition. In this work, we adopted a protein design strategy to study the functional switch
of KaiA during this process. We wanted to design a KaiA protein that could be reversibly switched off and on with light, but unfortunately, we did not fully achieve this goal in this work. Our design strategy gave us light regulated KaiA constructs, but the constructs did not work well in the in vitro reconstitution of the KaiABC system as expected. However, our data indicated that the N-terminal domain of KaiA is an indispensable player in the function switching of KaiA. Most interestingly, our data indicated that when KaiC and LOV2-Ja3-KaiA180C were co-incubated, KaiC was locked in a state prohibiting its re-phosphorylation. Furthermore, this locked state could not be rescued by the wild-type KaiA or KaiB. Combining our data, a likely explanation is once LOV2-Ja3KaiA180C became auto-inhibited, it did not fully release from KaiC; meanwhile, our previous study demonstrated that conformational changes provide stronger KaiA-KaiC interaction [6], so fresh KaiA could not bind with hypo-phosphorylated KaiC and stimulate its phosphorylation. With these data and recent studies from others [8,11] and our group [7], we proposed a possible model (Fig. 3E) of the KaiABC system, in which KaiB’s function is not limited to
Please cite this article as: J. Li et al., The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.072
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Fig. 3. The functional exploration of LOV2-Ja3-KaiA180C. (A) LOV2-Ja3-KaiA180C and KaiC were co-incubated in cycled dark/light environments to test if KaiC would have oscillated phosphorylation/de-phosphorylation changes. The green box indicates the time window for constant light. (B) KaiC was incubated at 30 C for 12 h to de-phosphorylate, then KaiA proteins (the wild-type KaiA, or LOV2-Ja3-KaiA180C) were added to stimulate KaiC’s phosphorylation. (C) LOV2-Ja3-KaiA180C and KaiC were co-incubated for 24 h, during which KaiC phosphorylated and then de-phosphorylated. At the indicated time point (24 h), additional KaiA proteins (the wild-type KaiA, or LOV2-Ja3-KaiA180C) were added to the solution to test if the fresh KaiA proteins would stimulate the phosphorylation of KaiC. (D) LOV2-Ja3-KaiA180C, KaiB and KaiC were co-incubated to test if KaiB could induce the rephosphorylation of KaiC. (E) A working model of the co-operation of KaiA and KaiB during the clock oscillation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
antagonize KaiA and induce the de-phosphorylation of KaiC, but it also chaperones the auto-inhibited KaiA and helps restore KaiA’s active conformation. During this process, KaiB is like a “hand” whereas the N-terminal domain of KaiA is a “latch”, so that KaiB flips the handle to turn off and turn on KaiA’s function sequentially (unidirectionally), which guarantees the unidirectional running of the clock in addition to the sequential phosphorylation/dephosphorylation of KaiC [27]. Recent studies showed that non-TTFL circadian clocks universally exist in many organisms, and the role of non-TTFL circadian clocks is indispensable for robust circadian rhythms [24,25]. Besides being an important model for non-TTFL circadian clocks, the KaiABC circadian clock is also intriguing for studying biological oscillators and protein-protein interaction [28]. We hope our work will help elucidating the function and mechanism of the KaiABC circadian clock. Declaration of competing interest The authors declare no competing financial interests. Acknowledgements We would like to thank Dr. Carl Johnson (Vanderbilt University) for generously providing the plasmids containing the Kai proteins. S. L. was supported by the grants from National Natural Science Foundation of China (31670768, 31971150), Hubei Provincial Science and Technology Department (2019CFA069), Wuhan Science and Technology Bureau of China (2018060401011319), and the
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Please cite this article as: J. Li et al., The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.072