Centrin is a conserved calcium-binding protein that plays an important role in diverse cellular biological processes such as ciliogenesis, gene expression, DNA repair and signal transduction. In Trypanosoma brucei, TbCentrin4 is mainly localized in basal bodies and bi-lobe structure, and is involved in the processes coordinating karyokinesis and cytokinesis. In the present study, we solved the solution structure of TbCentrin4 using NMR (nuclear magnetic resonance) spectroscopy. TbCentrin4 contains four EF-hand motifs consisting of eight α-helices. Isothermal titration calorimetry experiment showed that TbCentrin4 has a strong Ca2+ binding ability. NMR chemical shift perturbation indicated that TbCentrin4 binds to Ca2+ through its C-terminal domain composed of EF-hand 3 and 4. Meanwhile, we revealed that TbCentrin4 undergoes a conformational change and self-assembly induced by high concentration of Ca2+. Intriguingly, localization of TbCentrin4 was dispersed or disappeared from basal bodies and the bi-lobe structure when the cells were treated with Ca2+in vivo, implying the influence of Ca2+ on the cellular functions of TbCentrin4. Besides, we observed the interactions between TbCentrin4 and other Tbcentrins and revealed that the interactions are Ca2+ dependent. Our findings provide a structural basis for better understanding the biological functions of TbCentrin4 in the relevant cellular processes.

Introduction

Centrins (also known as caltractins) are evolutionarily conserved components of centrosomes, which are characterized at the microtubule-organizing center (MTOC). They are essential proteins for various regulatory functions in eukaryotes, including mRNA export, DNA repair, signal transduction, duplication and segregation of centrosome/basal bodies, and cell division [1,2]. Centrins have been identified in various organisms, from unicellular flagellates to vertebrates. Centrin was first identified in the unicellular green algae, where it forms filamentous structures extending from the nucleus–basal body connector to the distal contractile fibers [3,4]. In Saccharomyces cerevisiae, one centrin (Cdc31) was identified. Cdc31 localizes to the spindle pole body (SPB) and plays an important role in the cell cycle-dependent SPB duplication [57]. In addition, Cdc31 binding to Sfi1 is thought to play a role in the initiation of budding SPB duplication [8] and microtubule severing [9]. In human, three isoforms of centrins were identified [1012]. HsCentrin1 and HsCentrin2 are involved in centrosome/basal body segregation, and HsCentrin3 plays an essential role in centrosome/basal body duplication [12].

So far, several structures of centrins and centrin complexes have been reported [7,8,1316]. Similar to calmodulins (CaM), structure of centrin contains four ‘helix-loop-helix’ EF-hand motifs that are potential Ca2+-binding sites and fold into two independent and structurally similar domains, C-terminal domain (CTD) and N-terminal domain (NTD). The two independent domains are separated by a linker region (an α-helix or a short loop), shaping like a dumbbell [3,17]. The CTD of centrin is highly conserved [8,18,19]. The unique feature of centrin different from CaM and other Ca2+-binding proteins is that centrin contains an unstructured stretch of 20–25 residues in its NTD, which is not conserved among centrins [16,17]. As a Ca2+ sensor, the function of centrin is mediated by different binding partners and regulated by Ca2+ levels [20]. Centrin responds to cellular Ca2+ influx by selectively binding to Ca2+ through four EF-hand motifs. The binding of Ca2+ to centrin promotes the conformational change from a closed state to an open state, exposing the hydrophobic surface to interact with its partners for signaling [17,21,22]. In general, the CTD of centrin is able to bind Ca2+ with a higher affinity and is primarily responsible for its interaction with partners [8,20,23,24], whereas the NTD typically binds Ca2+ and its partners with a lower affinity [8,16,24].

Trypanosoma brucei is a protozoan parasite of the order Kinetoplastida, which causes sleeping sickness in human and nagana in cattle in sub-Saharan Africa. In comparison with three centrins found in human and only one centrin in Saccharomyces cerevisiae, five centrin isoforms have been identified in T. brucei [25,26], which implies that the centrins in T. brucei may have more multiple biological roles. Though the five isoforms share some sequence similarity, they are on utterly different branches of the evolutionary tree of centrins and calmodulins [27]. Moreover, they are not grouped in any of the four typical types of mammalian centrins identified previously [28]. TbCentrin1 (Tb927.04.2260) localizes to the flagellar basal body and is involved in organelle segregation and cytokinesis [29]. TbCentrin2 (Tb927.08.1080) is enriched within the bi-lobe structure and flagellar basal body, which is critical for Golgi duplication and segregation [25]. TbCentrin3 (Tb927.10.8710) is a flagellate protein located in the flagellum and regulates flagellar motility by forming a complex with an inner-arm dynein TbIAD5-1 [30]. TbCentrin4 (Tb927.07.3410) is present on both the basal bodies and the bi-lobe structure and is necessary for coordinating karyokinesis and cytokinesis [27]. Moreover, the interactions between TbCentrin2 and TbCentrin4 suggest that the cell cycle-dependent TbCentrin4 expression is able to regulate the abundance of TbCentrin2 [31], which implies that they may co-ordinate the process of nuclear and cell division. So far, there is little information about TbCentrin5 (Tb927.11.13900).

Although several studies focusing on the functions of centrins from T. brucei (TbCentrins) have been carried out, little is known about their structural information and the effects on their structure and function by Ca2+. Illustrating the structure and function of TbCentrins is of vital importance to better understand how TbCentrins participate in the relevant cellular process. In the present study, we determined the solution structure of TbCentrin4 and investigated its interactions with Ca2+ and other centrins.

Materials and methods

Cloning, expression and protein purification

Full-length genes encoding TbCentrin4 were amplified by PCR from the genome of T. brucei and cloned into the vector pET-22b (+) (Novagen), which provided the recombinant protein 6× His tag at the C-terminus. The recombinant vector was transformed into host Escherichia coli BL21 (DE3) for expression. The culture was fermented at 37°C to OD600 of 0.8 and induced with 0.5 mM IPTG (isopropyl-β-d-thiogalactoside) at 16°C for 20 h. The cells were harvested and suspended in buffer containing 20 mM Tris and 500 mM NaCl at pH 7.0. After sonication and centrifugation, the supernatant of lysed cells was collected and purified with a Ni-chelating column. The eluted protein was further purified by gel filtration column Sephadex G-75 (GE Healthcare). 15N,13C-labeled TbCentrin4 protein was prepared in the same way, except that LB medium was replaced by M9 medium containing 0.5 g/l 99% 15N-labeled ammonium chloride as nitrogen source and 2.5 g/l 99% 13C-glucose as carbon source. NMR samples contained 0.5 mM TbCentrin4, 20 mM phosphate (pH 6.5), 200 mM sodium chloride, 2 mM EDTA and 2 mM DTT in either 90% H2O/10% D2O or 100% D2O.

NMR experiments and structure calculation

All NMR data were collected at 293 K on a Bruker DMX600 spectrometer. A set of standard triple-resonance spectra including 1H-15N HSQC, HNCO, HNCACB, CBCA(CO)NH, CC(CO)NH, HC(CO)NH, (H)CCH-TOCSY and HCCH-COSY was recorded for backbone and side chain assignments. NMR data were processed with NMRpipe, NMRDraw software and SPARKY3 [32,33]. NOE distance restraints were obtained from 3D 15N- and 13C-edited NOESY spectra acquired with a mixing time of 130 ms. Backbone dihedral angle restraints were calculated by TALOS program and structures were calculated by CYANA program [34,35]. The final 20 lowest-energy structures were analyzed with MOLMOL [36]. The Ramachandran plot was analyzed with PROCHECK online [37].

NMR chemical shift perturbation

To investigate the interactions between TbCentrin4 and Ca2+/TbCentrin5, NMR chemical shift perturbation was performed. The sample of 15N-labeled TbCentern4 titrated with Ca2+ was mixed with EDTA which was then removed by gel filtration column Sephadex G-75 (GE Healthcare). TbCentern4 was extensively dialyzed against a buffer containing 20 mM phosphate (pH 6.5), 200 mM sodium chloride and 2 mM DTT. After that, 0.5 mM 15N-labeled TbCentrin4 was titrated with an increasing concentration of Ca2+ of 5 µM, 0.5 mM, 1 mM, 2 mM and 5 mM. Samples of 15N-labeled TbCentern4 titrated with TbCentrin5 were treated as above, except that they were dialyzed with or without 5 µM CaCl2. The increasing molar ratio of TbCentrin5/TbCentrin4 was 0.5, 1.0 and 2.0. A series of 1H-15N HSQC spectra were acquired at each concentration of Ca2+ or TbCentrin5 for analysis. The observed chemical shift change (Δδ) for each backbone amide was measured as the weighted average of the proton and nitrogen chemical shift changes using the equation: Δδ = [(Δδ2HN + Δδ2N/25)/2]1/2 [38].

Isothermal titration calorimetry

Isothermal titration calorimetry measurements were performed on iTC200 (GE Healthcare) at 20°C to investigate the Ca2+-binding capacity and the interactions between TbCentrin4 and TbCentrin1/2/3/5. Samples of TbCentern4 titrated with Ca2+ were mixed with EDTA and then desalted with a HiPrep 26/10 desalting column (GE Healthcare). TbCentrin4 and calcium ions were equilibrated in the same buffer containing 20 mM Tris–HCl (pH 7.0) and 200 mM NaCl. Approximately 50 µM TbCentrin4 in cell was titrated against 1.0 mM CaCl2 from syringe. About 2 µl of CaCl2 was injected into 0.2 ml of TbCentrin4 at 120 s intervals. Samples of TbCentrin1/2/3/5 titrated with Tbcentrin4 were purified with a Ni-chelating column and a Sephadex column G-75 (GE Healthcare). In total, 50 µM TbCentrin1/2/3/5 in the cell was titrated against 0.5 mM TbCentrin4 from the syringe with the same sample volume and rotate speed. The data collected was analyzed by MicroCal LLC ITC software (MicroCal).

GST pull-down assay

GST pull-down assay was performed to identify the interactions between Tbcentrin4 and other centrins from T. brucei. TbCentrin4 was expressed with a triple HA tag and TbCentrin1/2/3/5 were fused with a GST tag. TbCentrin1/2/3/5 fused by GST were incubated with prepared glutathione sepharose beads (GE Healthcare) at room temperature for 15 min and washed three times with PBS containing 0.1% Triton X-100. Input protein (TbCentrin4-3HA) was then incubated with the beads 30 min and washed as before. The target proteins were washed down with 50 mM GSSH (pH 7.2) and detected by SDS–PAGE and western blotting.

Circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy was performed on a Chirascan qCD spectrophotometer (Applied Photophysics) at 293 K. Far-UV CD spectra were recorded between 200 and 250 nm using a 0.5 mm path length cuvette. CD spectra were run with a step-resolution of 1 nm, an integration time of 0.5 s and a bandwidth of 1 nm. The spectra were averaged over three scans and corrected by subtraction of the buffer signal. In total, 5 µM TbCentrin4 with 2 mM EGTA or 0.05/0.5/5 mM CaCl2 was dissolved in a buffer containing 10 mM Tris and 150 mM NaCl at pH 7.0.

Trypanosoma cell culture and in situ tagging of TbCentrin4

The wild-type procyclic Lister 427 strain was cultivated at 28°C in Cunningham's medium supplemented with 10% fetal bovine serum. The full-length TbCentrin4 cDNA was fused to a modified pN-PURO-PTP vector [39] with a triple HA epitope tag in the N-terminus. The resulting construct was linearized and electroporated into the 427 cell line for in situ tagging. Successful transfectants were selected under 2 µg/ml puromycin and cloned by limiting dilution. Expression of TbCentrin4-3HA fusion protein was verified by western blotting.

Saponin permeabilization

Cells (∼1.5 × 107) expressing TbCentrin4-3HA were collected by centrifugation at 2000×g for 5 min, and the resuspended pellet was washed twice in resuspended buffer (20 mM Tris–HCl and 150 mM NaCl, pH 7.0). Next, cells were resuspended in a buffer (20 mM Tris–HCl, 150 mM NaCl, 0.05 mg/ml saponin and 5 mM CaCl2, pH 7.0) and were incubated for 5 min. Subsequently, cells were centrifuged and washed three times in resuspended buffer. The control was performed as above except 5 mM CaCl2 was replaced by 2 mM EDTA.

Immunofluorescence microscopy

Immunofluorescence staining was carried out according to our published procedures [40]. In brief, cells stably expressing TbCentrin4-3HA were harvested. Cells were fixed with 4% paraformaldehyde, then washed and blocked with blocking buffer (PBS with 1% BSA and 0.5% Triton X-100). After that, cells were incubated with the primary antibody and secondary antibody sequentially. The slides were then stained with DAPI (4′,6-diamidino-2-phenylindole) and examined using an inverted microscope (Model IX73, Olympus) with a cooled CCD (charge coupled device) camera (Model DP80, Olympus). Images were analyzed by softWoRx Explorer and ImageJ.

Results and discussion

Sequence analysis of TbCentrin4

TbCentrin4, with 149 residues in length, is the shortest one of five centrins in T. brucei. The sequence alignment of centrin family proteins was performed using ClustalW2 and ESPript 3.0 [41,42]. The alignment showed that TbCentrin4 shares 30–50% sequence identity with other four centrins from T. brucei and ∼40% with human centrins (Figure 1A). Compared with human Centrin1, Centrin2 and Centrin3 which share high sequence identity (53–84%) and highly similar sequence length (172 aa, 172 aa and 167 aa, respectively), the centrins from T. brucei display more diversity in the sequence. The sequence diversity is prominently present in the amino-terminal domain with the first 20 residues. In contrast with most centrins containing ∼170 residues, TbCentrin4 has only 149 residues, lacking the N-terminal 20 residues present in other centrins. TbCentrin4 consists of eight α-helices, predicted by Jpred 4 online [43], and four EF-hand motifs, analyzed by SMART online [44] (Figure 1A), with conserved residues located mainly in EF-hand domain regions.

Sequence alignments and solution structure of TbCentrin4.

Figure 1.
Sequence alignments and solution structure of TbCentrin4.

(A) Multiple sequence alignments of TbCentrin4 with other centrins. Tb, T. brucei; Hs, Homo sapiens. Identical residues are shaded in red box and conserved residues are colored in red. The secondary structure of TbCentrin4 determined is also included at the top of the sequence alignments. The domain architecture of TbCentrin4 is also included at the below of the sequence alignments. (B) Cartoon representation of the lowest target function structure of TbCentrin4. (C) Electrostatic surface diagram of the lowest-energy conformation of TbCentrin4 (red, negative; blue, positive; white, neutral). The two representations are shown from two different orientations (180° apart), and the view in (C) is oriented similar to that in (B).

Figure 1.
Sequence alignments and solution structure of TbCentrin4.

(A) Multiple sequence alignments of TbCentrin4 with other centrins. Tb, T. brucei; Hs, Homo sapiens. Identical residues are shaded in red box and conserved residues are colored in red. The secondary structure of TbCentrin4 determined is also included at the top of the sequence alignments. The domain architecture of TbCentrin4 is also included at the below of the sequence alignments. (B) Cartoon representation of the lowest target function structure of TbCentrin4. (C) Electrostatic surface diagram of the lowest-energy conformation of TbCentrin4 (red, negative; blue, positive; white, neutral). The two representations are shown from two different orientations (180° apart), and the view in (C) is oriented similar to that in (B).

Solution structure of TbCentrin4

Recombinant TbCentrin4 containing a C-terminal His-tag (HHHHHH) was expressed and purified. To ensure the total removal of Ca2+, TbCentrin4 was pretreated with EDTA or EGTA. The solution structure of TbCentrin4 was determined by a set of NMR spectra. 1H-15N HSQC spectrum of TbCentrin4 is shown in Supplementary Figure S1. The resonances disperse broadly and evenly, suggesting that the protein folds well and presents as a monomer. Five hundred structures of TbCentrin4 were calculated. In total, 1868 NOE distance restraints and 291 dihedral angle restraints were included in the structure calculation. The chemical shifts of the resonances have been deposited into the Biological Magnetic Resonance Data Bank (ID: 36 179). The 20 lowest energy structures have been deposited in the Protein Data Bank (PDB ID: 5ZOR). Cartoon of the best representative structure and its electrostatic surface are shown in Figure 1B and C, respectively. The RMSD (root-mean-square deviation) of the well-defined regions in the secondary structures of the 20 structures was 0.82 Å for the backbone and 1.10 Å for the heavy atoms. The Ramachandran plot was analyzed by PROCHECK to check the quality of the structure. The data show that 93.1% of the residues are in the most favored region, 6.9% in the allowed region and none in the disallowed region. Structural parameters of the solution structure of TbCentrin4 are summarized in Table 1.

Table 1
NMR structural statistics of TbCentrin4
NMR constraints in the structure calculation 
 Intraresidue 489 
 Sequential (|i − j| = 1) 566 
 Medium range (|i − j| < 5) 627 
 Long range (|i − j| ≥ 5) 186 
 Hydrogen bonds 118 
 Total distance constraints 1868 
 Dihedral angle constraints 291 
Residual violations 
 CYANA target functions (Å) 2.02 × 10−2 ± 9.21 × 10−2 
 Minimum (Å) 2.02 × 10−2 
 Maximum (Å) 2.06 × 10−2 
NOE upper distance constraint violations 
 Average (Å) 0.03 ± 0.00 
 Minimum (Å) 0.02 
 Maximum (Å) 0.03 
 RMS 0.0009 ± 0.0000 
 Number > 0.2 Å 0 ± 0 
NOE lower distance constraint violations 
 Average (Å) 0.01 ± 0.00 
 Minimum (Å) 0.01 
 Maximum (Å) 0.01 
 RMS 0.0018 ± 0.0001 
 Number > 0.2 Å 0 ± 0 
Dihedral angle constraint violations 
 Average (Å) 0.36 ± 0.00 
 Minimum (Å) 0.36 
 Maximum (Å) 0.37 
 RMS 0.0258 ± 0.0002 
 Number > 5 Å 0 ± 0 
van der Waals violations 
 Average (Å) 0.05 ± 0.00 
 Minimum (Å) 0.05 
 Maximum (Å) 0.05 
 Sum 0.3 ± 0.0 
 Number > 0.2 Å 0 ± 0 
Average structural RMSD to the mean co-ordinates (Å) 
 Secondary structure backbone atoms1 0.82 
 Secondary structure heavy atoms1 1.10 
 All backbone atoms2 0.86 
 All heavy atoms2 1.11 
Ramachandran statistics, % of residues 
 Most favored regions 93.1% 
 Additionally allowed regions 6.9% 
 Generously allowed regions 0.0% 
 Disallowed regions 0.0% 
Global quality scores (raw/Z score) 
 Verify3D 0.58/−0.83 
 MolProbity clash score 29.75/−4.39 
G-factors 
 Phi–psi 0.36 
 Dihedrals 0.11 
 Mainchain 0.70 
 Overall 0.34 
Planar groups 
 Within limits 100% 
 Highlighted 0.0% 
Model contents 
 Total no. of residues 149 
 BMRB accession number 36 179 
 PDB ID code 5ZOR 
NMR constraints in the structure calculation 
 Intraresidue 489 
 Sequential (|i − j| = 1) 566 
 Medium range (|i − j| < 5) 627 
 Long range (|i − j| ≥ 5) 186 
 Hydrogen bonds 118 
 Total distance constraints 1868 
 Dihedral angle constraints 291 
Residual violations 
 CYANA target functions (Å) 2.02 × 10−2 ± 9.21 × 10−2 
 Minimum (Å) 2.02 × 10−2 
 Maximum (Å) 2.06 × 10−2 
NOE upper distance constraint violations 
 Average (Å) 0.03 ± 0.00 
 Minimum (Å) 0.02 
 Maximum (Å) 0.03 
 RMS 0.0009 ± 0.0000 
 Number > 0.2 Å 0 ± 0 
NOE lower distance constraint violations 
 Average (Å) 0.01 ± 0.00 
 Minimum (Å) 0.01 
 Maximum (Å) 0.01 
 RMS 0.0018 ± 0.0001 
 Number > 0.2 Å 0 ± 0 
Dihedral angle constraint violations 
 Average (Å) 0.36 ± 0.00 
 Minimum (Å) 0.36 
 Maximum (Å) 0.37 
 RMS 0.0258 ± 0.0002 
 Number > 5 Å 0 ± 0 
van der Waals violations 
 Average (Å) 0.05 ± 0.00 
 Minimum (Å) 0.05 
 Maximum (Å) 0.05 
 Sum 0.3 ± 0.0 
 Number > 0.2 Å 0 ± 0 
Average structural RMSD to the mean co-ordinates (Å) 
 Secondary structure backbone atoms1 0.82 
 Secondary structure heavy atoms1 1.10 
 All backbone atoms2 0.86 
 All heavy atoms2 1.11 
Ramachandran statistics, % of residues 
 Most favored regions 93.1% 
 Additionally allowed regions 6.9% 
 Generously allowed regions 0.0% 
 Disallowed regions 0.0% 
Global quality scores (raw/Z score) 
 Verify3D 0.58/−0.83 
 MolProbity clash score 29.75/−4.39 
G-factors 
 Phi–psi 0.36 
 Dihedrals 0.11 
 Mainchain 0.70 
 Overall 0.34 
Planar groups 
 Within limits 100% 
 Highlighted 0.0% 
Model contents 
 Total no. of residues 149 
 BMRB accession number 36 179 
 PDB ID code 5ZOR 
1

Residues in secondary structures: 4–17, 26–36, 43–53, 63–75, 80–90, 100–109, 116–127 and 136–147.

2

Obtained for residues 4–14.

TbCentrin4 displays an overall fold comprising eight α-helices: α1 (residues 4–17), α2 (residues 26–36), α3 (residues 43–53), α4 (residues 63–75), α5 (residues 80–90), α6 (residues 100–109), α7 (residues 116–127) and α8 (residues 136–147). α1 and α2, α3 and α4, α5 and α6, α7 and α8 form four EF-hand motifs, respectively. Noticeably, α1 and α4, α2 and α3, α5 and α8, α6 and α7 are approximately parallel to each other, respectively. The overall structure of TbCentrin4 can be divided equally into two independent globular domains, NTD containing EF-hand 1 and 2 and CTD containing EF-hand 3 and 4. The two independent domains are connected by a flexible loop. Four EF hands pack against each other and are linked by loops of several residues. Each domain contains a pair of EF-hand motifs, which creates a hydrophobic pocket that is usually the Ca2+-binding site.

Structural comparison of TbCentrin4 with other CaM and centrin proteins

The structure of TbCentrin4 was submitted to DALI [45] to search for its similar structures. The proteins with the highest structural similarity to TbCentrin4 are those of CaM or centrin family members. The highest similar four are as follows: myosin regulatory light chain from Physarum polycephalum (PpMRLC) (PDB ID: 2BL0) [46], which is a CaM-like protein, Troponin C from Oryctolagus cuniculus (OcTnC) (PDB ID: 1TN4) [47], Calcineurin from Bos taurus (BtCaM) (PDB ID: 2F2P) [48] and Centrin1 from Mus musculus (MmCentrin1) (PDB ID: 5D43) [15]. The Cα RMSD between TbCentrin4 and PpMRLC, OcTnC, BtCaM and MmCentrin1 are 4.1, 2.8, 3.0 and 3.4 Å, with corresponding Z-scores of 9.1, 8.3, 8.1 and 8.0, respectively. Sequence alignment demonstrated that all these proteins share many highly conserved residues (Figure 2A). Compared with MmCentrin1, TbCentrin4 loses the first 20 residues in the N-terminus, more similar to CaM proteins (Figure 2A).

Structural comparison of TbCentrin4 with other CaM and centrin members.

Figure 2.
Structural comparison of TbCentrin4 with other CaM and centrin members.

(A) Sequence alignments of TbCentrin4, PpMRLC, OcTnC, BtCaM and MmCentrin1. Tb, Trypanosoma brucei; Pp, Physarum polycephalum; Oc, Oryctolagus cuniculus; Bt, Bos taurus; Mm, Mus musculus. Identical residues are shaded in red box and conserved residues are colored in red. The secondary structures of TbCentrin4 and MmCentrin1 are also included at the top and the below of the sequence alignments, respectively. Significant differences on the secondary structure are labeled with red star. (B) Structural comparison of TbCentrin4 with PpMRLC (PDB ID: 2BL0), OcTnC (PDB ID: 1TN4), BtCaM (PDB ID: 2F2P) and MmCentrin1 (PDB ID: 5D43). Red represents the structural variation linking NTD and CTD, blue represents the structural variation linking two α-helices of every EF-hand in the CTD and magenta represents the structural variation linking two α-helices of every EF-hand in the NTD.

Figure 2.
Structural comparison of TbCentrin4 with other CaM and centrin members.

(A) Sequence alignments of TbCentrin4, PpMRLC, OcTnC, BtCaM and MmCentrin1. Tb, Trypanosoma brucei; Pp, Physarum polycephalum; Oc, Oryctolagus cuniculus; Bt, Bos taurus; Mm, Mus musculus. Identical residues are shaded in red box and conserved residues are colored in red. The secondary structures of TbCentrin4 and MmCentrin1 are also included at the top and the below of the sequence alignments, respectively. Significant differences on the secondary structure are labeled with red star. (B) Structural comparison of TbCentrin4 with PpMRLC (PDB ID: 2BL0), OcTnC (PDB ID: 1TN4), BtCaM (PDB ID: 2F2P) and MmCentrin1 (PDB ID: 5D43). Red represents the structural variation linking NTD and CTD, blue represents the structural variation linking two α-helices of every EF-hand in the CTD and magenta represents the structural variation linking two α-helices of every EF-hand in the NTD.

TbCentrin4 and other four proteins all consist of four EF-hands, which form NTD and CTD. Although TbCentrin4 adopts a similar fold compared with CaM or centrin members from other species, some significant differences were observed (Figure 2B). The most significant difference lies in the length of α4. The α4 of TbCentrin4 is much shorter than that in OcTnC, BtCaM and MmCentrin1, resulting in more compact fold of TbCentrin4. Besides, no β-strand is present in the structure of TbCentrin4, while other proteins contain one or two β-strands.

For better understanding the structural characteristic of TbCentrin4, the structure of TbCentrin4 was compared with those of other centrin family members (Supplementary Figure S2) [8,1416]. Similarly, all these centrins have two independent domains each constituted by a pair of EF-hand motifs, indicating that centrins are structurally conserved in the evolution. Meanwhile, a notable difference lies in the length of helix α4. Similar to yeast centrin (ScCdc31) [8], the fold of TbCentrin4 is more compact than other centrins [1416], resulting from the much shorter helix α4. In other centrins such as MmCentrin1 [15], the long helix α4 pulls NTD and CTD far away from each other, therefore leading to a relatively open fold. This structural variation implies the existence of different centrin subfamilies in the evolution and may be the basis for centrins to participate in different biological processes.

Tbcentirn4 binds to Ca2+ through its CTD containing EF-hand 3 and 4

Calcium ion (Ca2+) is an important second messenger in regulating biological processes [22,49]. To investigate the Ca2+-binding property of TbCentirn4, ITC was performed with an endothermic reaction (Figure 3A). The thermodynamic parameters of the interaction between Ca2+ and TbCentrin4 (pretreated with EDTA) are shown in Table 2, which were fitted with a two-site sequential binding model. The affinities (Kd) of the two sites were 3.1 and 38 µM, respectively, suggesting that TbCentrin4 has a high affinity for Ca2+ binding and one site has a higher affinity than the other one. The binding reaction is an entropically (ΔS) favorable and enthalpically (ΔH) unfavorable process, indicating that Ca2+ binding results in the release of water molecules by an increase in solvent entropy.

The Ca2+ binding of TbCentirn4.

Figure 3.
The Ca2+ binding of TbCentirn4.

(A) Saturated titration of TbCentrin4 with Ca2+ was measured by ITC. (B) Overlay of 1H-15NHSQC spectra of 15N-labeled TbCentrin4 in the absence (red) or presence (green) of 5 µM CaCl2. (C) Chemical shift changes of TbCentrin4 induced by 5 µM CaCl2. The horizontal solid line represents the calculated average chemical shift perturbations. Asterisk represents missing resonances that are not visible in the spectra; p represents proline. (D) Sequence alignments of CTD of TbCentrin4, CrCentrin, ScCdc31, HsCentrin2 and MmCentrin1. Tb, Trypanosoma brucei; Cr, Chlamydomonas reinhardtii; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens; Mm, Mus musculus. Identical residues are shaded in red box and conserved residues are colored in red. The secondary structure of CTD of TbCentrin4 is included at the top of the sequence alignments. Arrows represent the residues involving in interaction with calcium ions.

Figure 3.
The Ca2+ binding of TbCentirn4.

(A) Saturated titration of TbCentrin4 with Ca2+ was measured by ITC. (B) Overlay of 1H-15NHSQC spectra of 15N-labeled TbCentrin4 in the absence (red) or presence (green) of 5 µM CaCl2. (C) Chemical shift changes of TbCentrin4 induced by 5 µM CaCl2. The horizontal solid line represents the calculated average chemical shift perturbations. Asterisk represents missing resonances that are not visible in the spectra; p represents proline. (D) Sequence alignments of CTD of TbCentrin4, CrCentrin, ScCdc31, HsCentrin2 and MmCentrin1. Tb, Trypanosoma brucei; Cr, Chlamydomonas reinhardtii; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens; Mm, Mus musculus. Identical residues are shaded in red box and conserved residues are colored in red. The secondary structure of CTD of TbCentrin4 is included at the top of the sequence alignments. Arrows represent the residues involving in interaction with calcium ions.

Table 2
Thermodynamic parameters for Ca2+ binding to TbCentrin4 obtained by ITC
 Ka (M−1Kd (µM) ΔH (kcal/mol) ΔS (cal/mol/K) 
Site 1 3.18 × 105 ± 4.63 × 104 3.1 4.40 × 103 ± 32 40.2 
Site 2 2.63 × 104 ± 4.37 × 103 38 1.34 × 103 ± 282 30.7 
 Ka (M−1Kd (µM) ΔH (kcal/mol) ΔS (cal/mol/K) 
Site 1 3.18 × 105 ± 4.63 × 104 3.1 4.40 × 103 ± 32 40.2 
Site 2 2.63 × 104 ± 4.37 × 103 38 1.34 × 103 ± 282 30.7 

To further examine which residues in TbCentrin4 are involved in the interaction with Ca2+, chemical shift perturbation experiment was performed with increasing concentration of Ca2+. When 0.5 mM TbCentrin4 was titrated with a low concentration (5 µM) of Ca2+, obvious chemical shift changes were observed in the EF-hand 3 and 4 of the CTD of TbCentrin4 (Figure 3B,C). These residues with significant chemical shift change may interact directly with Ca2+ or be affected by Ca2+ binding. The chemical shift perturbation of residues in EF-hand 4 is more significant than those in EF-hand 3, suggesting higher affinity in EF-hand 4 and lower affinity in EF-hand 3, consistent with our ITC analysis which indicates that one site has a higher affinity for Ca2+ than the other one.

The two-site model of TbCentrin4 binding to Ca2+ is similar to that of HsCentrin2 [16], Cdc31 [8] and CrCentrin [14], in which one centrin molecule binds two Ca2+ by its EF-hand 3 and 4 in the CTD. EF-hand adopts a ‘helix-loop-helix’ fold that forms a single Ca2+-binding site. Calcium ion interacts with residues within the turn-loop region. Sequence alignment of CTD of well-characterized centrins whose structures have been determined demonstrates that the residues involved in Ca2+ binding [8,1416,50] are conserved (Figure 3D). Based on the analysis from chemical shift perturbation, the Ca2+- binding site I of TbCentrin4 might contain Asp91, Asp93, Lys95, Lys97 and Asn102 in EF-hand 3 and the Ca2+-binding site II might contain Asp129, Asp131, Glu133 and Glu138 in EF-hand 4. The residues of TbCentrin4 involved in direct interaction with Ca2+ are located at the loop of EF-hand where the nonpolar amino acids create a hydrophobic pocket and the polar amino acids interact with Ca2+ through electrostatic interactions.

Tbcentrin4 undergoes simultaneously conformational changes and self-assembly in a high concentration of Ca2+

1H-15N HSQC spectra of 15N-labeled TbCentrin4 titrated with a high concentration of Ca2+ (2 mM) were also recorded (Figure 4A). The results demonstrated that a majority of peaks in both CTD and NTD were observed with significant shift upon the addition of Ca2+. Moreover, the peaks did not disperse broadly and evenly anymore after the titration. Instead, the peaks overlapped heavily and some peaks were missing. The phenomenon indicated that TbCentrin4 may undergo conformational changes and/or self-assembly under a high concentration of Ca2+.

Conformational changes and self-assembly of TbCentrin4 induced by a high concentration of Ca2+.

Figure 4.
Conformational changes and self-assembly of TbCentrin4 induced by a high concentration of Ca2+.

(A) The 1H,15N-HSQC spectra of TbCentrin4 in the absence (red) or presence of 2 mM Ca2+ (blue). (B) Far-UV CD spectra of 5 µM TbCentrin4 in the presence of 2 mM EGTA (black), 0.05 mM CaCl2 (red), 0.5 mM CaCl2 (blue) and 5 mM CaCl2 (green), respectively. The spectra were averaged over three scans and corrected by subtraction of the buffer signal. (C) Gel filtration chromatography of TbCentrin4 without (black) or with 5 mM CaCl2 (red).

Figure 4.
Conformational changes and self-assembly of TbCentrin4 induced by a high concentration of Ca2+.

(A) The 1H,15N-HSQC spectra of TbCentrin4 in the absence (red) or presence of 2 mM Ca2+ (blue). (B) Far-UV CD spectra of 5 µM TbCentrin4 in the presence of 2 mM EGTA (black), 0.05 mM CaCl2 (red), 0.5 mM CaCl2 (blue) and 5 mM CaCl2 (green), respectively. The spectra were averaged over three scans and corrected by subtraction of the buffer signal. (C) Gel filtration chromatography of TbCentrin4 without (black) or with 5 mM CaCl2 (red).

CD was used to characterize the content of secondary structural elements in TbCentrin4 and determine whether any conformational changes occurred upon Ca2+ binding. The far-UV CD spectra of TbCentrin4 with or without Ca2+ (pretreated with EGTA) are similar in shape, with two troughs at 208 and 222 nm, typical of a protein with a high amount of helical structure (Figure 4B). This suggests that the global secondary structure of TbCentrin4 is not changed by Ca2+. An obvious increase in molar ellipticity (monitored at 208 and 222 nm) was observed on the addition of Ca2+. This increase is similar to that in the case of the well-studied Ca2+ sensor CaM [51,52]. In that case, three-dimensional structures determined in the absence or presence of Ca2+ revealed that the increase in molar ellipticity after binding to Ca2+ resulted from the reorganization of the disposition of the helices within the EF-hand domains. Therefore, binding of Ca2+ through the loops of EF-hands of TbCentrin4 might induce a local conformational change of TbCentrin4 in a similar way.

To detect whether TbCentrin4 will self-assemble induced by the high concentration of Ca2+, gel filtration chromatography was performed (Figure 4C). TbCentrin4 without Ca2+ (pretreated with EDTA) was eluted from a Sephadex G-75 gel filtration column as a single peak at the volume of 72 ml with an apparent molecular mass of 18 kDa, which is very close to the monomeric molecular mass of 16.5 kDa. TbCentrin4 with 5 mM CaCl2 was eluted from a Sephadex G-75 gel filtration column in three peaks. The first peak appeared at the volume of 53 ml, suggesting the existence of TbCentrin4 polymer. The second peak appeared at the volume of 63 ml with an apparent molecular mass of 33 kDa, suggesting the existence of TbCentrin4 dimer. The third peak appeared at the volume of 72 ml where TbCentrin4 monomer should be. The gel filtration chromatography showed that a high concentration of Ca2+ will make TbCentrin4 self-assembly to dimer and polymer.

The localization of TbCentrin4 was indistinct or disappeared from basal bodies and bi-lobe structure when the cells were treated with Ca2+in vivo

TbCentrin4 is located in basal bodies and bi-lobe structure reported previously [27]. TbCentrin4 with a triple HA epitope tag in the N-terminus was used to investigate its subcellular localization. The results demonstrated that TbCentrin4 is localized in basal bodies and bi-lobe structure as expected (Figure 5A). To further investigate the change of the localization of TbCentrin4 upon Ca2+ binding, cells were permeabilized with saponin to ensure the entry of Ca2+ or EDTA (as a control without Ca2+) into the cytoplasm. The results showed that the localization of TbCentrin4 was indistinct or disappeared from basal bodies and bi-lobe structure when cells were treated with 5 mM CaCl2 (Figure 5B). As the control, the localization of TbCentrin4 did not change when cells were treated with 2 mM EDTA (Figure 5C). Our study indicated that the binding of calcium ion will affect the localization of TbCentrin4 in vivo.

Subcellular localization of TbCentrin4.

Figure 5.
Subcellular localization of TbCentrin4.

The localization of TbCentrin4-3HA was examined in paraformaldehyde-fixed intact cells. Cells were stained with anti-HA antibody for TbCentrin4-3HA (green) and DAPI for DNA (blue). 1N1K, 1N2K and 2N2K cells were tabulated, respectively. (A) Cells were not permeabilized with saponin. (B) Cells were permeabilized with saponin supplemented with 5 mM CaCl2. (C) Cells were permeabilized with saponin supplemented with 2 mM EDTA. Scale bars: 5 µm.

Figure 5.
Subcellular localization of TbCentrin4.

The localization of TbCentrin4-3HA was examined in paraformaldehyde-fixed intact cells. Cells were stained with anti-HA antibody for TbCentrin4-3HA (green) and DAPI for DNA (blue). 1N1K, 1N2K and 2N2K cells were tabulated, respectively. (A) Cells were not permeabilized with saponin. (B) Cells were permeabilized with saponin supplemented with 5 mM CaCl2. (C) Cells were permeabilized with saponin supplemented with 2 mM EDTA. Scale bars: 5 µm.

A previous study has reported that a high concentration of calcium can induce a decrease in the centriole diameter, the shortening of the intercentriolar link that connects centrioles [53] and basal body reorientation on an isolated basal apparatus of Chlamydomonas reinhardtii [54]. In other cases, such as necrosis, autophagy, apoptosis and death, the concentration of calcium will increase instantly [5560]. Therefore, the local conformational change of TbCentrin4 and its self-assembly, accompanied by the indistinctness and disappearance of TbCentrin4 from basal bodies and bi-lobe structure induced by the high concentration of calcium, might be related to those biological processes.

Tbcentrin4 interacts with other centrins and the interactions are Ca2+-dependent

Five centrin isoforms have been identified in T. brucei [25,26]. The previous work has identified the interactions between TbCentrin4 and TbCentrin2 [31]. To further investigate the interactions between TbCentrin4 and other four centrins, GST pull-down assay was performed. TbCentrin4-HA and GST-fused TbCentrin1, 2, 3, 5 were used in the GST pull-down assay. The results demonstrated that TbCentrin4 interacts with TbCentrin1, TbCentrin2 and TbCentrin5 but not TbCentrin3 (Figure 6A). To further confirm it, ITC analysis of TbCentrin1, 2, 3, 5 titrated with TbCentrin4 was performed (Figure 6B). The result verified the interactions between TbCentrin4 and TbCentrin1, 2, 5. The dissociate constants (Kd values) of TbCentrin4 interacting with TbCentrin1, TbCentrin2 and TbCentrin5 were 100, 120 and 10 µM, respectively.

Interactions between TbCentrin4 and other centrins without EDTA treatment.

Figure 6.
Interactions between TbCentrin4 and other centrins without EDTA treatment.

(A) Western blotting with anti-HA antibody to detect TbCentrin4-3HA pulled down by GST-TbCentrin1/2/3/5. (B) ITC of TbCentrin1/2/3/5 titrated with TbCentrin4.

Figure 6.
Interactions between TbCentrin4 and other centrins without EDTA treatment.

(A) Western blotting with anti-HA antibody to detect TbCentrin4-3HA pulled down by GST-TbCentrin1/2/3/5. (B) ITC of TbCentrin1/2/3/5 titrated with TbCentrin4.

It needs to be mentioned that in these experiments, EDTA was not added to remove remained Ca2+ in TbCentrins. Since Ca2+ may be copurified with TbCentrins during the expression and purification procedures, it is necessary to ensure complete removal of Ca2+ by the treatment of EDTA to investigate the effect of Ca2+ on the interactions between TbCentrin4 and other centrins. TbCentrin1–5 were first mixed with EDTA to remove Ca2+. ITC analysis of TbCentrin1, 2, 3, 5 titrated with TbCentrin4 was then performed without Ca2+ (Supplementary Figure S3). In the absence of Ca2+, TbCentrin4 appeared no interaction with TbCentrin1, 2, 3, 5. The results indicated that the interactions between TbCentrin4 and TbCentrin1, 2, 5 are Ca2+-dependent.

To further verify the effect of Ca2+ on the interactions between TbCentrin4 and other centrins, chemical shift perturbation of 15N-labeled TbCentrin4 (pretreated with EDTA) titrated with increasing TbCentrin5 (which has the highest affinity to TbCentrin4) was performed in the absence or in the presence of 5 µM Ca2+. Although no significant shift of resonance was detected in the 1H-15N HSQC spectra of TbCentrin4 titrated with TbCentrin5 (Figure 7A,B), obvious decrease in peak intensity was observed in the presence of Ca2+ (Figure 7C,D). Both in the absence and presence of Ca2+, the intensities of overall peaks of TbCentrin4 decreased similarly, which should be due to the dilution by the addition of TbCentrin5. However, most of the peak intensity of the residues in EF-hand 3 and EF-hand 4 of TbCentrin4 became weaken more distinct than those of EF-hand 1 and EF-hand 2 in the presence of Ca2+ (Figure 7C), suggesting that these residues might be important for the interactions. All these results indicated that TbCentrin4 interacts with other centrins and the interactions are Ca2+-dependent.

The effect of Ca2+ on the interactions between TbCentrin4 and TbCentrin5.

Figure 7.
The effect of Ca2+ on the interactions between TbCentrin4 and TbCentrin5.

Chemical shift perturbation of 15N-labeled TbCentrin4 titrated with TbCentrin5 in the presence (A) or absence (B) of 5 µM Ca2+. 1H-15NHSQC spectra of TbCentrin4 with (red) or without (blue) TbCentrin5. The peak intensity ratio of TbCentrin4 titrated with TbCentrin5 is quantified in the presence (C) or absence (D) of 5 µM Ca2+. The molar ratio of TbCentrin5/TbCentrin4 is 2. The horizontal solid line represents the mean value. Asterisk represents missing resonances that are not visible in the spectra; p represents proline.

Figure 7.
The effect of Ca2+ on the interactions between TbCentrin4 and TbCentrin5.

Chemical shift perturbation of 15N-labeled TbCentrin4 titrated with TbCentrin5 in the presence (A) or absence (B) of 5 µM Ca2+. 1H-15NHSQC spectra of TbCentrin4 with (red) or without (blue) TbCentrin5. The peak intensity ratio of TbCentrin4 titrated with TbCentrin5 is quantified in the presence (C) or absence (D) of 5 µM Ca2+. The molar ratio of TbCentrin5/TbCentrin4 is 2. The horizontal solid line represents the mean value. Asterisk represents missing resonances that are not visible in the spectra; p represents proline.

It is noteworthy that the Ca2+-binding sites are also located on the EF-hand 3 and 4. One explanation is that Ca2+ binding induces the local conformational change of TbCentrin4, which results in the exposure of more hydrophobic region of TbCentrin4 to interact with itself or other centrins. Owing to these interactions, TbCentrin4 is able to form different complexes with different centrins to accommodate various cellular biological processes by a Ca2+-dependent way in T. brucei.

In conclusion, we determined the solution structure of TbCentrin4 by NMR spectroscopy. TbCentrin4 binds to Ca2+ through its CTD and undergoes the conformational change, self-assembly and localization change. Besides, TbCentrin4 is able to interact with other TbCentrins and the interactions are regulated by Ca2+. Our study will shed light on understanding the structure and function of centrins in T. brucei.

Abbreviations

     
  • CaM

    calmodulin

  •  
  • CD

    circular dichroism

  •  
  • CTD

    C-terminal domain

  •  
  • DAPI

    4′,6-diamidino-2-phenylindole

  •  
  • ITC

    isothermal titration calorimetry

  •  
  • MTOC

    microtubule organizing center

  •  
  • NMR

    nuclear magnetic resonance

  •  
  • NTD

    N-terminal domain

  •  
  • PDB

    Protein Data Bank

  •  
  • RMSD

    root-mean-square deviation

  •  
  • SPB

    spindle pole body

Author Contribution

X.T., F.S. and S.L. designed the research. F.S., K.Y. and J.Z. performed the experiments. F.S., S.L. and X.T. analyzed the data. F.S. and X.T. wrote the paper. X.Z. and C.X. discussed and gave advice on the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 31270780 and U1332137 (to X.T.), 31500601 (to S.L.) and 31570737 (to C.X.)].

Acknowledgments

We are grateful to Ziyin Li of the University of Texas Health Science Center at Houston for providing the wild-type T. brucei 427 procyclic-form cells and plasmids (pN-PURO-PTP) used for in situ tagging. We also thank F. delaglio and A. Bax for providing NMRPipe and NMRDraw, T.D. Goddard and D. Kneller for Sparky, and R. Koradi and K. Wuthrich for MOLMOL.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Salisbury
,
J.L.
,
Baron
,
A.T.
and
Sanders
,
M.A.
(
1988
)
The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells
.
J. Cell Biol.
107
,
635
641
2
Salisbury
,
J.L.
(
1995
)
Centrin, centrosomes, and mitotic spindle poles
.
Curr. Opin. Cell Biol.
7
,
39
45
3
Salisbury
,
J.L.
,
Baron
,
A.
,
Surek
,
B.
and
Melkonian
,
M.
(
1984
)
Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle
.
J. Cell Biol.
99
,
962
970
4
Schiebel
,
E.
and
Bornens
,
M.
(
1995
)
In search of a function for centrins
.
Trends Cell Biol.
5
,
197
201
5
Baum
,
P.
,
Furlong
,
C.
and
Byers
,
B.
(
1986
)
Yeast gene required for spindle pole body duplication: homology of its product with Ca2+-binding proteins
.
Proc. Natl Acad. Sci. U.S.A.
83
,
5512
5516
6
Spang
,
A.
,
Courtney
,
I.
,
Grein
,
K.
,
Matzner
,
M.
and
Schiebel
,
E.
(
1995
)
The Cdc31p-binding protein Kar1p is a component of the half bridge of the yeast spindle pole body
.
J. Cell Biol.
128
,
863
877
7
Ivanovska
,
I.
and
Rose
,
M.D.
(
2001
)
Fine structure analysis of the yeast centrin, Cdc31p, identifies residues specific for cell morphology and spindle pole body duplication
.
Genetics
157
,
503
518
PMID:
[PubMed]
8
Li
,
S.
,
Sandercock
,
A.M.
,
Conduit
,
P.
,
Robinson
,
C.V.
,
Williams
,
R.L.
and
Kilmartin
,
J.V.
(
2006
)
Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication
.
J. Cell Biol.
173
,
867
877
9
Salisbury
,
J.L.
(
2004
)
Centrosomes: Sfi1p and centrin unravel a structural riddle
.
Curr. Biol.
14
,
R27
R29
10
Lee
,
V.D.
and
Huang
,
B.
(
1993
)
Molecular cloning and centrosomal localization of human caltractin
.
Proc. Natl Acad. Sci. U.S.A.
90
,
11039
11043
11
Errabolu
,
R.
,
Sanders
,
M.A.
and
Salisbury
,
J.L.
(
1994
)
Cloning of a cDNA encoding human centrin, an EF-hand protein of centrosomes and mitotic spindle poles
.
J. Cell Sci.
107
,
9
16
PMID:
[PubMed]
12
Middendorp
,
S.
,
Paoletti
,
A.
,
Schiebel
,
E.
and
Bornens
,
M.
(
1997
)
Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevisiae CDC31 gene
.
Proc. Natl Acad. Sci. U.S.A.
94
,
9141
9146
13
Popescu
,
A.
,
Miron
,
S.
,
Blouquit
,
Y.
,
Duchambon
,
P.
,
Christova
,
P.
and
Craescu
,
C.T.
(
2003
)
Xeroderma pigmentosum group C protein possesses a high affinity binding site to human Centrin 2 and calmodulin
.
J. Biol. Chem.
278
,
40252
40261
14
Sosa Ldel
,
V.
,
Alfaro
,
E.
,
Santiago
,
J.
,
Narváez
,
D.
,
Rosado
,
M.C.
,
Rodríguez
,
A.
et al. 
(
2011
)
The structure, molecular dynamics, and energetics of centrin-melittin complex
.
Proteins
79
,
3132
3143
15
Kim
,
S.Y.
,
Kim
,
D.S.
,
Hong
,
J.E.
and
Park
,
J.H.
(
2017
)
Crystal structure of wild-type centrin 1 from Mus musculus occupied by Ca2+
.
Biochemistry
82
,
1129
1139
16
Thompson
,
J.R.
,
Ryan
,
Z.C.
,
Salisbury
,
J.L.
and
Kumar
,
R.
(
2006
)
The structure of the human Centrin 2-xeroderma pigmentosum group C protein complex
.
J. Biol. Chem.
281
,
18746
18752
17
Wiech
,
H.
,
Geier
,
B.M.
,
Paschke
,
T.
,
Spang
,
A.
,
Grein
,
K.
,
Steinkötter
,
J.
et al. 
(
1996
)
Characterization of green alga, yeast, and human centrins. Specific subdomain features determine functional diversity
.
J. Biol. Chem.
271
,
22453
22461
18
Gavet
,
O.
,
Alvarez
,
C.
,
Gaspar
,
P.
and
Bornens
,
M.
(
2003
)
Centrin4p, a novel mammalian centrin specifically expressed in ciliated cells
.
Mol. Biol. Cell
14
,
1818
1834
19
Vonderfecht
,
T.
,
Stemm-Wolf
,
A.J.
,
Hendershott
,
M.
,
Giddings
, Jr,
T.H.
,
Meehl
,
J.B.
and
Winey
,
M.
(
2011
)
The two domains of centrin have distinct basal body functions in Tetrahymena
.
Mol. Biol. Cell
22
,
2221
2234
20
Zhang
,
Y.
and
He
,
C.Y.
(
2012
)
Centrins in unicellular organisms: functional diversity and specialization
.
Protoplasma
249
,
459
467
21
Zhou
,
Q.
,
Gheiratmand
,
L.
,
Chen
,
Y.
,
Lim
,
T.K.
,
Zhang
,
J.
,
Li
,
S.
et al. 
(
2010
)
A comparative proteomic analysis reveals a new bi-lobe protein required for bi-lobe duplication and cell division in Trypanosoma brucei
.
PLoS ONE
5
,
e9660
22
Sanz
,
J.M.
,
Grecu
,
D.
and
Assairi
,
L.
(
2016
)
Ca2+ signaling and target binding regulations: calmodulin and centrin in vitro and in vivo
.
Bioenergetics, Open Access
5
,
1000144
23
Radu
,
L.
,
Durussel
,
I.
,
Assairi
,
L.
,
Blouquit
,
Y.
,
Miron
,
S.
,
Cox
,
J.A.
et al. 
(
2010
)
Scherffelia dubia centrin exhibits a specific mechanism for Ca2+-controlled target binding
.
Biochemistry
49
,
4383
4394
24
Veeraraghavan
,
S.
,
Fagan
,
P.A.
,
Hu
,
H.
,
Lee
,
V.
,
Harper
,
J.F.
,
Huang
,
B.
et al. 
(
2002
)
Structural independence of the two EF-hand domains of caltractin
.
J. Biol. Chem.
277
,
28564
28571
25
He
,
C.Y.
,
Pypaert
,
M.
and
Warren
,
G.
(
2005
)
Golgi duplication in Trypanosoma brucei requires Centrin2
.
Science
310
,
1196
1198
26
Berriman
,
M.
,
Ghedin
,
E.
,
Hertz-Fowler
,
C.
,
Blandin
,
G.
,
Renauld
,
H.
,
Bartholomeu
,
D.C.
et al. 
(
2005
)
The genome of the African trypanosome Trypanosoma brucei
.
Science
309
,
416
422
27
Shi
,
J.
,
Franklin
,
J.B.
,
Yelinek
,
J.T.
,
Ebersberger
,
I.
,
Warren
,
G.
and
He
,
C.Y.
(
2008
)
Centrin4 coordinates cell and nuclear division in T. brucei
.
J. Cell Sci.
121
,
3062
3070
28
Friedberg
,
F.
(
2006
)
Centrin isoforms in mammals. Relation to calmodulin
.
Mol. Biol. Rep.
33
,
243
252
29
Selvapandiyan
,
A.
,
Kumar
,
P.
,
Morris
,
J.C.
,
Salisbury
,
J.L.
,
Wang
,
C.C.
and
Nakhasi
,
H.L.
(
2007
)
Centrin1 is required for organelle segregation and cytokinesis in Trypanosoma brucei
.
Mol. Biol. Cell
18
,
3290
3301
30
Wei
,
Y.
,
Hu
,
H.
,
Lun
,
Z.-R.
and
Li
,
Z.
(
2014
)
Centrin3 in trypanosomes maintains the stability of a flagellar inner-arm dynein for cell motility
.
Nat. Commun.
5
,
4060
31
Wang
,
M.
,
Gheiratmand
,
L.
and
He
,
C.Y.
(
2012
)
An interplay between Centrin2 and Centrin4 on the bi-lobed structure in Trypanosoma brucei
.
Mol. Microbiol.
83
,
1153
1161
32
Delaglio
,
F.
,
Grzesiek
,
S.
,
Vuister
,
G.W.
,
Zhu
,
G.
,
Pfeifer
,
J.
and
Bax
,
A.
(
1995
)
NMRPipe: a multidimensional spectral processing system based on UNIX pipes
.
J. Biomol NMR
6
,
277
293
33
Goddard
,
T.
and
Kneller
,
D.
(
2004
)
SPARKY 3
,
University of California
,
San Francisco
34
Güntert
,
P.
,
Mumenthaler
,
C.
and
Wüthrich
,
K.
(
1997
)
Torsion angle dynamics for NMR structure calculation with the new program DYANA
.
J. Mol. Biol.
273
,
283
298
35
Shen
,
Y.
,
Delaglio
,
F.
,
Cornilescu
,
G.
and
Bax
,
A.
(
2009
)
TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts
.
J. Biomol. NMR
44
,
213
223
36
Koradi
,
R.
,
Billeter
,
M.
and
Wüthrich
,
K.
(
1996
)
MOLMOL: a program for display and analysis of macromolecular structures
.
J. Mol. Graph.
14
,
51
55
37
Laskowski
,
R.A.
,
MacArthur
,
M.W.
,
Moss
,
D.S.
and
Thornton
,
J.M.
(
1993
)
PROCHECK: a program to check the stereochemical quality of protein structures
.
J. Appl. Crystallogr.
26
,
283
291
38
Grzesiek
,
S.
,
Bax
,
A.
,
Clore
,
G.M.
,
Gronenborn
,
A.M.
,
Hu
,
J.-S.
,
Kaufman
,
J.
et al. 
(
1996
)
The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation of the binding surface for the SH3 domain of Hck tyrosine protein kinase
.
Nat. Struct. Mol. Biol.
3
,
340
39
Schimanski
,
B.
,
Nguyen
,
T.N.
and
Günzl
,
A.
(
2005
)
Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination
.
Eukaryot. Cell
4
,
1942
1950
40
Yang
,
X.
,
Wu
,
X.
,
Zhang
,
J.
,
Zhang
,
X.
,
Xu
,
C.
,
Liao
,
S.
et al. 
(
2017
)
Recognition of hyperacetylated N-terminus of H2AZ by TbBDF2 from Trypanosoma brucei
.
Biochem. J.
474
,
3817
3830
41
Gouet
,
P.
,
Courcelle
,
E.
,
Stuart
,
D.I.
and
Metoz
,
F.
(
1999
)
ESPript: analysis of multiple sequence alignments in PostScript
.
Bioinformatics
15
,
305
308
42
Larkin
,
M.A.
,
Blackshields
,
G.
,
Brown
,
N.
,
Chenna
,
R.
,
McGettigan
,
P.A.
,
McWilliam
,
H.
et al. 
(
2007
)
Clustal W and Clustal X version 2.0
.
Bioinformatics
23
,
2947
2948
43
Cuff
,
J.A.
,
Clamp
,
M.E.
,
Siddiqui
,
A.S.
,
Finlay
,
M.
and
Barton
,
G.J.
(
1998
)
JPred: a consensus secondary structure prediction server
.
Bioinformatics
14
,
892
893
44
Schultz
,
J.
,
Copley
,
R.R.
,
Doerks
,
T.
,
Ponting
,
C.P.
and
Bork
,
P.
(
2000
)
SMART: a web-based tool for the study of genetically mobile domains
.
Nucleic Acids Res.
28
,
231
234
45
Holm
,
L.
and
Sander
,
C.
(
1993
)
Protein structure comparison by alignment of distance matrices
.
J. Mol. Biol.
233
,
123
138
46
Debreczeni
,
J.É.
,
Farkas
,
L.
,
Harmat
,
V.
,
Hetényi
,
C.
,
Hajdú
,
I.
,
Závodszky
,
P.
et al. 
(
2005
)
Structural evidence for non-canonical binding of Ca2+ to a canonical EF-hand of a conventional myosin
.
J. Biol. Chem.
280
,
41458
41464
47
Houdusse
,
A.
,
Love
,
M.L.
,
Dominguez
,
R.
,
Grabarek
,
Z.
and
Cohen
,
C.
(
1997
)
Structures of four Ca2+-bound troponin C at 2.0 Å resolution: further insights into the Ca2+-switch in the calmodulin superfamily
.
Structure
5
,
1695
1711
48
Ye
,
Q.
,
Li
,
X.
,
Wong
,
A.
,
Wei
,
Q.
and
Jia
,
Z.
(
2006
)
Structure of calmodulin bound to a calcineurin peptide: a new way of making an old binding mode
.
Biochemistry
45
,
738
745
49
McCormack
,
J.
and
Denton
,
R.
(
1986
)
Ca2+ as a second messenger within mitochondria
.
Trends Biochem. Sci.
11
,
258
262
50
Charbonnier
,
J.B.
,
Renaud
,
E.
,
Miron
,
S.
,
Le Du
,
M.H.
,
Blouquit
,
Y.
,
Duchambon
,
P.
et al. 
(
2007
)
Structural, thermodynamic, and cellular characterization of human Centrin 2 interaction with xeroderma pigmentosum group C protein
.
J. Mol. Biol.
373
,
1032
1046
51
Martin
,
S.R.
and
Bayley
,
P.M.
(
1986
)
The effects of Ca2+ and Cd2+ on the secondary and tertiary structure of bovine testis calmodulin. A circular-dichroism study
.
Biochem. J.
238
,
485
490
52
Martin
,
S.R.
,
Lu
,
A.Q.
,
Xiao
,
J.
,
Kleinjung
,
J.
,
Beckingham
,
K.
and
Bayley
,
P.M.
(
1999
)
Conformational and metal-binding properties of androcam, a testis-specific, calmodulin-related protein from Drosophila
.
Protein Sci.
8
,
2444
2454
53
Moudjou
,
M.
,
Paintrand
,
M.
,
Vigues
,
B.
and
Bornens
,
M.
(
1991
)
A human centrosomal protein is immunologically related to basal body-associated proteins from lower eucaryotes and is involved in the nucleation of microtubules
.
J. Cell Biol.
115
,
129
140
54
Hayashi
,
M.
,
Yagi
,
T.
,
Yoshimura
,
K.
and
Kamiya
,
R.
(
1998
)
Real-time observation of Ca2+-induced basal body reorientation in Chlamydomonas
.
Cell Motil. Cytoskeleton
41
,
49
56
55
Pinton
,
P.
,
Giorgi
,
C.
,
Siviero
,
R.
,
Zecchini
,
E.
and
Rizzuto
,
R.
(
2008
)
Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis
.
Oncogene
27
,
6407
56
Wang
,
S.-H.
,
Shih
,
Y.-L.
,
Ko
,
W.C.
,
Wei
,
Y.-H.
and
Shih
,
C.-M.
(
2008
)
Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway
.
Cell. Mol. Life Sci.
65
,
3640
3652
57
Høyer-Hansen
,
M.
and
Jäättelä
,
M.
(
2007
)
Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium
.
Cell Death Differ.
14
,
1576
58
Orrenius
,
S.
,
Zhivotovsky
,
B.
and
Nicotera
,
P.
(
2003
)
Calcium: regulation of cell death: the calcium-apoptosis link
.
Nat. Rev. Mol. Cell Biol.
4
,
552
59
Decuypere
,
J.-P.
,
Bultynck
,
G.
and
Parys
,
J.B.
(
2011
)
A dual role for Ca2+ in autophagy regulation
.
Cell Calcium
50
,
242
250
60
Rizzuto
,
R.
,
Giorgi
,
C.
,
Romagnoli
,
A.
and
Pinton
,
P.
(
2008
)
Ca2+ signaling, mitochondria and cell death
.
Curr. Mol. Med.
8
,
119
130

Supplementary data