Identification and characterization of a novel ...

Biochemical and Biophysical Research Communications xxx (2018) 1e6

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Identification and characterization of a novel calmodulin binding site in Drosophila TRP C-terminus ZiLing Sun, YunHua Zheng, Wei Liu* Shenzhen Key Laboratory of Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, Guangdong, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 April 2018 Accepted 2 May 2018 Available online xxx

Transient receptor potential (TRP) channels are a group of essential cation channels involved in many important sensory signal transduction processes, such as light, temperature, tastes and pressure sensing. Drosophila TRP channel is the first discovered family member and plays important roles in phototransduction in Drosophila. Calmodulin (CaM), an important downstream effector of Ca2þ signal, was considered as a vital regulator of TRP activities. In this study, we discovered a novel Ca2þ dependent CaM binding site (TRP 783e862) in between the previously reported two calmodulin binding sites (CBSs). The isothermal titration calorimetry (ITC) and the size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) results showed that the dissociation constant (Kd) between TRP 783 e862 and Ca2þ-CaM is 0.10 ± 0.04 mM and their binding stoichiometry is 1:1. In addition, the shortest Ca2þ-CaM interaction region and core CaM binding sequences in TRP 783e862 were dissected by the boundary mapping and mutagenesis experiments. More interestingly, by comparing the circular dichroism (CD) spectra before and after Ca2þ-CaM binding, the TRP 783e862 fragment showed Ca2þ-CaM binding dependent secondary structure changes, indicating that the interaction between CaM and Drosophila TRP channel may have a conformational impact on TRP structure. In summary, by identifying and characterizing a novel CaM binding site in TRP C-terminus, our findings provided a biochemical and structural basis for further in vivo functional studies of Ca2þ-mediated TRP channel regulation through CaM/TRP interaction. © 2018 Elsevier Inc. All rights reserved.

Keywords: TRP channel Calmodulin Calmodulin binding site

1. Introduction Transient receptor potential (TRP) channels are a large family of Ca2þ-permeable cation channels, which are well known to mediate sensory transduction such as sensations of vision, pain, temperature, tastes, touch, etc [1]. So far, more than 30 TRP channels have been found, which can be classified into 7 sub-families: TRPC, TRPA, TRPV, TRPP, TRPM, TRPML and TRPN [2]. Among of them, Drosophila TRP, the ortholog of mammal TRPC sub-family, is the first discovered TRP family member. In 1969, Cosens et al. showed that the trp mutant fly only produced a transient receptor potential when exposed to light [3]. Decades later, the Trp gene was cloned by Montell et al. [4]. Soon after that, Hardie et al. found that it encoded a Ca2þ permeable channel [5]. Meanwhile, TRP-like (TRPL), a TRP homologous gene, was also identified and reported to participate in

* Corresponding author. E-mail address: [email protected] (W. Liu).

Drosophila photo-transduction cascade [6]. The TRP channel consists of a N-terminal intracellular ankyrin repeats, six-transmembrane helixes and a long intracellular C-terminal cytoplasmic tail, which contains a TRP box, KP repeats, DKDKK (PG/AD) repeat and PDZ binding motif (PBM) [7e9] (Fig. 1). Previous studies showed that Ca2þ played both negative and positive feedback roles on Drosophila TRP channels [10]. Calmodulin (calcium-modulated protein, CaM), an important downstream effector, was considered as a vital regulator in this process. In 1997, Montell et al. proved that TRP C-terminal 687e977 fragment could bind CaM in the presence of Ca2þ using overlay experiments [11]. Later, using the GST pull-down assay, the Drosophila TRP 723e754 fragment was demonstrated to be a CaM binding site (CBS) [12]. Recently, another CBS in Drosophila TRP channel, TRP 863e940 fragment, has been reported to bind to CaM in Ca2þ-dependent manner [13]. We wondered whether there would be other calmodulin binding sites (CBS) in between TRP 723e754 and TRP 863e940 (Fig. 1). In this study, by utilizing the Escherichia coli (E.coli) expression

https://doi.org/10.1016/j.bbrc.2018.05.007 0006-291X/© 2018 Elsevier Inc. All rights reserved.

Please cite this article in press as: ZiLing-g. Sun, et al., Identification and characterization of a novel calmodulin binding site in Drosophila TRP Cterminus, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.007

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Z. Sun et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e6

system, we expressed and purified the TRP fragments in between the previously discovered two CBSs (Fig. 1). We identified the TRP 783e862 fragment as a new Ca2þ-CaM binding site. The isothermal titration calorimetry (ITC) and the size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) results showed that the CaM bound to TRP 783e862 strongly (dissociation constant (Kd) ¼ 0.10 ± 0.04 mM) with 1:1 binding stoichiometry in Ca2þ-dependent manner. Further mapping experiments showed the TRP 813e862 fragment was the shortest Ca2þ-CaM binding region in TRP 783e863. In addition, based on sequence alignment analysis and the “Calmodulin Targets” database, we proposed a core CaM binding sequences in TRP 783e940 region, which fitted the 15-10 Ca2þ-CaM binding pattern. Consistent with our prediction, following mutagenesis analysis showed that LAtoEE and VItoEE mutations could eliminate the Ca2þ dependent CaM binding of TRP 783e862 fragment. Finally, the subtracted CD spectra showed that the Ca2þ-CaM binding could induce secondary structure changes of TRP 783e862 fragment, which indicating that Ca2þ-CaM binding may have a conformational impact on TRP structure and therefore the regulation of Drosophila TRP channel activities.

2. Materials and methods 2.1. Constructs and protein expression The cDNA encoding different fragments of TRP were amplified by polymerase chain reaction (PCR) from a full-length TRP cDNA template (Drosophila Genomics Resource Center, NCBI accession ID: NM_057420.4) and cloned into a modified pET vector [14] via BamHI/XhoI cutting sites. Since the amino acids of CaM is very conserved, the cDNA encoding the Drosophila CaM was generated from a Human CaM cDNA by standard PCR-based mutagenesis method (Q144T & A148S) and inserted into the modified pET vector. The protein expression plasmids were transformed into Rosetta (DE3) strains of E. Coli. Single colony was inoculated and grown to OD600 ¼ 0.6. Then 1 mM IPTG was added to induce the protein expression in 16 C for 18 h. Cells were harvested and re-suspended in the binding buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 5 mM imidazole) supplemented with 1 mM PMSF. The collected cells were crushed by a high pressure homogenizer (Union biotech, China). After centrifugation at 18,000 rpm for 30 min, the supernatant was purified by the Ni2þ-sepharose beads (GE Healthcare) and followed by a size exclusion chromatography (HiLoad 26/60 Superdex 200 prep grade, GE healthcare) with a buffer of 50 mM Tris (pH 7.8), 100 mM NaCl, 1 mM DTT, and 1 mM EDTA or 1 mM CaCl2. 3C protease was used to remove the Trx-His tag of CaM and TRP fragments. The CaM after cutting the Trx-His tag was further purified by an anion exchange chromatography (HiPrep Q FF 16/10, GE healthcare), while the TRP fragments after 3C cutting were

further purified by a cation exchange chromatography (Hi Prep SP HP 16/10, GE healthcare). 2.2. Isothermal titration calorimetry assay (ITC) ITC measurements were carried out on a MicroCal ITC200 calorimeter (Malvern, USA) at 25 C. The proteins were dissolved in the titration buffer containing 50 mM Tris-HCl (pH 7.8), 100 mM NaCl, 1 mM DTT, and 1 mM EDTA or 1 mM CaCl2. 300 mM CaM in the syringe was titrated into 30 mM different TRP fragments in the cell sequentially by injecting a 2 mL aliquot at each titration point with a time interval of 120s. The final titration data were analyzed by Origin7 and fitted with the one-site binding model. 2.3. Size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) Protein samples (100 ml, 100 mM) was injected into a Superdex 200 Increase 10/300 GL column (GE healthcare) equilibrated with a buffer containing 50 mM Tris-HCl (pH 7.8), 100 mM NaCl, 1 mM DTT, and 1 mM EDTA or 1 mM CaCl2. The AKTA chromatography system was coupled with a Dawn Heleos-II light scattering detector (Wyatt Technologies) and an Optilab-Rex refractive index monitor (Wyatt Technologies). Molecular weight analyses were performed by using the ASTRA (Wyatt Technologies) software. 2.4. Circular dichroism (CD) spectra measurements Circular dichroism spectra were obtained by using a Chirascan instrument (Applied Photophysics, UK) with a thermostated cell holder. Spectra were recorded from 200 to 260 nm with three repeats using a quartz cell of 0.5 mm light path. The final concentration of protein samples were 35 mM in buffers containing 50 mM Tris-HCl (pH 7.8), 100 mM NaCl, 1 mM DTT, with or without 1 mM CaCl2. Each CD spectrum was subtracted by the buffer blank first. The final subtracted CD spectra were obtained by subtracting the CD spectrum of CaM alone from the CD spectra of TRP 783e862/ CaM mixture with or without 1 mM CaCl2. 3. Results 3.1. A new CBS in between TRP 723e754 and TRP 863-940 Previous studies showed TRP 723e754 and TRP 863e940 could bind to CaM in Ca2þ-dependent manner [12,13]. We wonder whether there are other CBSs in between the TRP 723e754 and TRP 863e940 regions. Using E. coli expression system, we expressed and purified TRP 755e826 and TRP 783e862 fragments which covered the regions between the TRP 723e754 and TRP 863e940

Fig. 1. Domain organization of Drosophila TRP channel subunit. Schematic diagram of the domain organization of Drosophila TRP channel. Dashed boxes denote regions in the cytoplasm.



regions (Fig. 2A). ITC results showed that neither Ca2þ-CaM nor apo-CaM could interact with the TRP 755e826 fragment (Fig. 2B and C), while the TRP 783e862 fragment bound to Ca2þ-CaM in Ca2þ-dependent manner (Kd ¼ 0.1 ± 0.04 mM) with 1:1 binding stoichiometry (Fig. 2D). On the other hand, apo-CaM did not interact with the TRP 783e862 fragment (Fig. 2E). Therefore, we successfully identified a novel Ca2þ-CaM binding site in the region between TRP 723e754 and TRP 863e940 region. 3.2. SEC-MALS confirmed 1:1 stoichiometry between TRP 783e862 and Ca2þ-CaM In order to further verify the binding stoichiometry between TRP 783e862 and Ca2þ-CaM, we used the SEC-MALS method to measure the molecular weight of TRP 783e862/Ca2þ-CaM complex. In the presence of 1 mM CaCl2, the mixture of Ca2þ-CaM and TRP 783e862 had a smaller elution volume than Ca2þ-CaM and TRP

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783e862 alone, suggesting that Ca2þ-CaM could interact with TRP 783e862 (Fig. 2F). On the contrary, in the presence of 1 mM EDTA, the mixture of apo-CaM and TRP 783e862 did not show elution volume shift, indicating the apo-CaM did not bind to TRP 783e862 (Fig. 2G). In addition, the measured molecular weight of Ca2þ-CaM/ TRP 783e862 complex was 38.8 ± 0.4 kDa (Fig. 2F, red line), which fitted well with the theoretical molecular weight of 1:1 stoichiometry complex (40.2 kDa) and further confirmed the binding stoichiometry obtained from the ITC experiments. 3.3. Mapping the minimal Ca2þ-CaM binding region in TRP 783862 To narrow down the Ca2þ-CaM binding region in TRP 783e862, based on the sequence conservation analysis and secondary structure prediction (http://bioinf.cs.ucl.ac.uk/psipred/) (Fig. 3A) [15], several truncated TRP fragments were expressed and purified

Fig. 2. A new CBS in between the TRP 723e754 and TRP 863e940. A. The region in between the TRP 723e754 and TRP 863e940 was divided into two fragments: TRP 755e826 and TRP 783e862. B. TRP 755e826 could not interact with Ca2þ-CaM. C. TRP 755e826 could not bind to apo-CaM. D. Binding affinity of the interaction between the TRP 783e862 and Ca2þ-CaM was measured by ITC; Kd ¼ 0.1 ± 0.04 mM and N ¼ 1.01 ± 0.02. E. TRP 783e862 could not interact with apo-CaM. F. SEC-MALS results showing that Trx-TRP 783e862 and Ca2þ-CaM form a 1:1 complex. G. SEC-MALS showing that Trx-TRP 783e862 could not interact with apo-CaM. n. d. means undetectable.


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Fig. 3. Sequence analysis and identification of the minimal boundary and core Ca2þ-CaM binding residues in TRP783-940. A. Sequence analysis of TRP 783e862: logo map of conserved residues with secondary structure prediction (the overall height of the stack indicates the sequence conservation at that position, while the relative height of symbols within the stack indicates the relative frequency of each amino acid at that position). B. Sequence alignment of CBSs in Drosophila TRP, TRPL and Human TRPC1: the predicted 1-5-10 Ca2þ-CaM binding pattern was highlighted in red shadow; positive charged amino acids were highlighted in yellow shadow. The positions of the mutated core Ca2þ-CaM binding residues (LA and VI) is labeled in red boxes. C. Summary of the dissociation constants derived from the ITC experiments to deduce the minimal interaction region and identify the core Ca2þ-CaM binding residues in the TRP 783e862 region. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

to test their Ca2þ-CaM bindings using the ITC assay (Fig. 3C). The results showed that after truncating the first non-conserved 20 amino acids in the N-terminus, the binding affinity between the TRP 802e862 fragment and Ca2þ-CaM was similar to the TRP 783e862 fragment (Fig. 3C & Fig. S1A). Further truncating the Nterminus to TRP 813e862 did not change the binding affinity either (Fig. 3C & Fig. S1B). However, when the N-terminal boundary was shortened to TRP 825e862, it turned out that the binding affinity significantly decreased (Fig. 3C & Fig. S1C). The synthesized TRP 822e862 peptide also showed a weak binding to Ca2þ-CaM (Fig. 3C & Fig. S1D), indicating the 813e822 region of TRP, which also highly conserved in the sequence alignment, was important for the Ca2þCaM binding. On the other hand, we also tried to narrow down the

C-terminal boundary of TRP 783e862. The results showed that truncating the C-terminal 7 residues (TRP 783e855) significantly decreased the binding affinity (Fig. 3C & Fig.S1E). Therefore, in our experimental settings, the minimal Ca2þ-CaM binding region in TRP 783e862 was mapped to TRP 813e862. 3.4. Critical Ca2þ-CaM binding residues in TRP 783-862 Based on the sequence alignment and prediction by calmodulin target database, TRP 832e859 as predicted to be the core Ca2þ-CaM binding region with high score (http://calcium.uhnres.utoronto.ca/) [16]. We analyzed the sequence of TRP 832e859 (SGRDIFSSLAKVIGRKKTQKGDKDWNAI), and found that it fitted well with the 1-5-



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Fig. 4. Ca2þ-CaM binding induced secondary structure change of TRP 783e862. A. Interaction with Ca2þ-CaM induced significant helical structure formation of TRP 783e862, showed by the subtracted CD spectrum of TRP 783e862 when mixing with Ca2þ-CaM in 1:1 ratio (colored in red); The control CD spectrum of TRP 783e862 alone in 1 mM Ca2þ is colored in green. B. Apo-CaM did not induce secondary structure change of TRP 783e862, showed by the subtracted CD spectrum of TRP 783e862 when mixed with apo-CaM in 1:1 ratio (colored in red); the control CD spectrum of TRP 783e862 alone is labeled in green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

10 Ca2þ-CaM binding mode (Fig. 3B) [17]. Since Ca2þ-CaM tends to interact with the positive charged and hydrophobic residues of its targets [18], we tried to mutate the core hydrophobic residues to glutamate to test our hypothesis. As shown in Fig. 3C & Fig. S2A, consistent with our prediction, mutations of Leu840 and Ala841 to Glu could abolish the binding between Ca2þ-CaM and TRP 783e862. While mutations of Val843 and Ile844 to Glu could also greatly weaken the binding affinity (Fig. 3C & Fig. S2B). 3.5. Ca2þ-CaM binding induced secondary structure change of TRP 783-862 To test whether Ca2þ-CaM binding will induce secondary structure change of TRP 783e862, the CD spectra before and after Ca2þCaM binding were recorded with or without 1 mM CaCl2. In presence of 1 mM Ca2þ, adding 1:1 stoichiometric Ca2þ-CaM induced helical structure formation of TRP 783e862 fragment, compared to TRP 783e862 fragment alone (Fig. 4A). Meanwhile, the subtracted CD spectra of TRP 783e862 in 1:1 mixture of apo-CaM showed no significant secondary structure change compared to TRP 783e862 alone (Fig. 4B). Our results showed that Ca2þ-CaM could induce secondary structure change of TRP 783e862 in Ca2þ-dependent manner. 4. Discussion TRP channels play important roles in Drosophila phototransduction cascade. However, the gating mechanisms of Drosophila TRP channel still remain unclear. Since calcium signal regulates TRP channels in positive and negative directions [10] and its important downstream effector, CaM, was also reported to interact with TRP C-terminus, it would be necessary to identify and characterize the interaction between CaM and the CBSs in TRP channel. According to the sequence alignment analysis (Fig.S3, inner circle), we noticed that Drosophila TRP N-terminus, transmembrane region, C-terminal 670e940 fragment and the last 14 residues were extremely conserved. In 2001, Tang et al. reported that GST-CaM could pull down TRP 723e754 (CBS1) in the presence of Ca2þ [12]. Recently, Zheng et al. discovered TRP 863e940 as a new CBS in TRP C-terminus and TRP 940e1275 did not interact with CaM [13]. We wondered whether there are other undiscovered CBSs in between TRP 723e754 and TRP 863e940 fragments, which are quite conserved in the sequence alignment analysis (Fig. S3, inner circle). Indeed, by expressing and purifying the TRP 755e826 and TRP 783e862 fragments (Fig. 2A), we successfully identified a

novel CBS in this region. Both ITC and SEC-MALS assays proved that CaM interact with TRP 783e862 in a Ca2þ-dependent manner with 1:1 stoichiometry. Further mapping and mutagenesis experiments showed that TRP 813e862 was the minimal Ca2þ-CaM binding region and mutating the critical residues in the predicted 1-5-10 Ca2þ-CaM binding region eliminated the interaction. In addition, we also found that Ca2þ-CaM could induce helical formation of TRP 783e862 fragment by using the subtracted CD spectra. There are some cases showing that CaM binding will induce a conformational rearrangement and regulate the activities of the target proteins, such as CaMKII and Ca2þ-activated potassium channels [19e21]. We proposed this property may have an impact on the conformation change of Drosophila TRP channels and therefore mediate the activity regulation of TRP channel. Taken the previous findings together, we conclude that there are three Ca2þ-dependent CBSs in TRP cytoplasmic C-terminal tail, as shown in Fig.S3. Such kind of multiple interactions between TRP Cterminus and Ca2þ-CaM may play important roles in the Ca2þdependent positive and negative feedback regulations of TRP channels [10]. In the future, based on the mutations discovered in this study, performing the electrophysiology experiments in transgenic flies will be helpful to test the roles of the interactions between TRP and CaM in Ca2þ-dependent positive and negative regulations of TRP channels. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31670765), the National Basic Research Program of China (973 Program) (2014CB910204), the National Key Research and Development Program (2016YFA0501900), the Natural Science Foundation of Guangdong Province (2016A030312016), and Shenzhen Basic Research Grants (JCYJ20170411090807530, JCYJ20160427185712266 and JCYJ20160229153100269). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.05.007. Transparency document Transparency document related to this article can be found


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