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PROTEIN STRUCTURE REPORT

Solution structure and backbone dynamics of Calsensin, an invertebrate neuronal calcium-binding protein

Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA

Reprint requests to: Jørgen Johansen, Department of Biochemistry, Biophysics, and Molecular Biology, 3156 Molecular Biology Building, Iowa State University, Ames, IA 50011, USA; e-mail: jorgen{at}iastate.edu; fax: (515) 294-4858.

(RECEIVED February 15, 2005; FINAL REVISION April 10, 2005; ACCEPTED April 11, 2005)

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Calsensin is an EF-hand calcium-binding protein expressed by a subset of peripheral sensory neurons that fasciculate into a single tract in the leech central nervous system. Calsensin is a 9-kD protein with two EF-hand calcium-binding motifs. Using multidimensional NMR spectroscopy we have determined the solution structure and backbone dynamics of calcium-bound Calsensin. Calsensin consists of four helices forming a unicornate-type four-helix bundle. The residues in the third helix undergo slow conformational exchange indicating that the motion of this helix is associated with calciumbinding. The backbone dynamics of the protein as measured by ¹⁵N relaxation rates and heteronuclear NOEs correlate well with the three-dimensional structure. Furthermore, comparison of the structure of Calsensin with other members of the EF-hand calcium-binding protein family provides insight into plausible mechanisms of calcium and target protein binding.

Keywords: Calsensin; calcium-binding proteins; EF-hand; NMR structure; dynamics; helix–loop–helix; nervous system

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051412605.

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Intracellular calcium concentration regulates a variety of cellular processes including neurite extension, cell motility, cell-cycle progression, cell proliferation, and apoptosis (Schafer and Heizmann 1996; Berridge et al. 1998; Donato 2003). Many of these signal transduction events are mediated by members of the EF-hand family of calcium-binding proteins via interaction with target proteins in a calcium-dependent manner (Schafer and Heizmann 1996). The EF-hand family of calcium-binding proteins can be classified as sensor or buffer proteins based on their function (Zimmer et al. 1995; Ikura 1996). The buffer proteins like Calbindin D_9K maintain calcium homeostasis by regulating the intracellular calcium concentration (Ikura 1996). On the other hand, sensor proteins such as S100B and CaM undergo a conformational change upon calcium binding, thereby altering their affinity for target proteins like caldesmon, tau annexins, and various kinases (Schafer and Heizmann 1996; Donato 2003). EF-hand calcium-binding proteins have been implicated in a variety of pathological diseases including Alzheimer’s disease, Down syndrome, and inflammatory disorders (Griffin et al. 1998). Thus, solving the structure of these proteins will be valuable for elucidating their functions.

We have previously cloned and characterized a small 9-kD neuronal EF-hand Ca²⁺-binding protein, Calsensin (Briggs et al. 1995). Calsensin is expressed in a subset of peripheral sensory neurons fasciculating into a single axon tract in the leech central nervous system (Briggs et al. 1993, 1995). The molecular features of Calsensin and its restricted expression in the nervous system are consistent with the hypothesis that it may participate in protein-complex mediated calcium-dependent signal transduction events in growth cones and axons (Briggs et al. 1995). It has become increasingly clear that changes in intracellular calcium levels can modulate axon fasciculation and growth cone motility (Hong et al. 2000; Zheng 2000) and previous studies have directly implicated calcium-binding proteins in growth cone guidance in Drosophila (VanBerkum and Goodman 1995). For these reasons we have undertaken a structural study of Calsensin, determining the structure of calciumbound Calsensin under reducing condition using multidimensional NMR spectroscopy.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
3D solution structure of Calsensin
In order to determine the 3Dsolution structure of Calsensin, sequence-specific resonance assignments were performed according to standard protocols (Wuthrich 1986) using 3D ¹⁵N-edited TOCSY and NOESY as well as 3D HNCACB, CBCACONH CCONH, and HCCH-TOCSY (Cavanagh 1995). All experiments were performed at 298 K with purified Calsensin from cell cultures grown in the presence of 1 mM CaCl₂ and resuspended in 50 mM sodium phosphate buffer (pH6.0) containing 75mMNaCl, 2mMDTT and 0.02%NaN₃. These conditions generated highly reproducible NMR spectra from eight independent purification and sample preparations. The backbone amide resonances were assigned for all but six N-terminal and three C-terminal amino acids. The 513 intra- and 1016 interresidue NOE assignments were obtained by analyzing 2D NOESY as well as 3D ¹⁵N-edited and ¹³C-edited NOESY spectra. The aromatic resonances were assigned based on 2D DQF-COSY and 2D NOESY data. The ³J_NH-H scalar coupling constants from HNHA data were used to obtain the angle constraints according to the Karplus equation (Wuthrich 1986). The angles for the remaining residues and the angles were obtained using TALOS (Cornilescu et al. 1999). Hydrogen bond restraints were introduced corresponding to slowly exchanging amide protons observed in the deuterium exchange data. A total of 1529 NOE, 44 hydrogen bond (there are two distance restraints for each hydrogen bond), and 78 dihedral angle constraints (Table 1) were used for final calculations with CNS. The 20 lowest energy structures have no distance violations greater than 0.4 Å and angle violations, greater than 5°, and the RMSDs from the experimental constraints and idealized covalent geometry are low. The pairwise RMSD as well as RMSD to the mean structure of these structures were relatively small (Table 1). Furthermore, the backbone dihedral angles of the majority of residues in the minimum average structure (85.1%) fall inside the most favorable regions of the Ramachandran plot (Table 1).

NH-H

View larger version (72K): [in this window] [in a new window]   Figure 1. (A) Overlay of the 20 lowest energy structures of Calsensin. The structures were superimposed using all the backbone residues in the secondary structural elements and rendered using MOLMOL. The N-terminal residues M1-K6 and C-terminal Q81- K83 were not assigned due to lack of sequential NOEs. All the helices are well-defined except for H3, which shows chemical exchange and higher RMSD as compared to the mean structure. (B) Surface plot of Calsensin rendered using MOLMOL. The regions colored in blue have positive electrostatic potential, those colored in red have negative electrostatic potential, and the regions in white are nonpolar. The hydrophobic residues in the helices 2, 3, and 4 as well as in the hinge region are exposed to the surface. (C) Stereo view of the energy minimized average ribbon structure of Calsensin. The labeling of the secondary structural elements follows the standard nomenclature used for EF-hand calcium-binding proteins. (D) Alignment of Calsensin with other members of the EF-hand family of calcium binding proteins. The sequence of Calsensin was aligned with CaM, Plastin-1 and with the members of S100 and polcalcin families. The alignment was generated using AlignX software of the VectorNTI Suite from Invitrogen. The residues in Calsensin that are identical are highlighted in yellow (polcalcin family) or cyan (other EF-hand members), while similar residues are shown in red. The two highly conserved aspartic acid residues are shown in red and highlighted in gray.

Comparison of Calsensin with other EF-hand calcium-binding proteins
The highest sequence identity between Calsensin and other members of the EF-hand superfamily is in the calcium-binding loops (Fig. 1D). Sequence alignment further shows that the calcium-binding loops of Calsensin are most similar to those of two members of the polcalcin family of pollen EF-hand calciumbinding proteins (Verdina et al. 2002; Neudecker et al. 2004). Although Calsensin can form dimers via oxidation of cysteine residues (data not shown), it is monomeric in solution under reducing conditions. Furthermore, Calsensin, unlike the members of S100 family (Drohat et al. 1998; Vallely et al. 2002), does not appear to form noncovalent dimers under experimental conditions. Bet v 4 exists as a monomer whereas another member of the polcalcin family, Phl p 7, revealed a domain-swapped dimer structure (Verdina et al. 2002; Neudecker et al. 2004). In general, the EF-hand calcium-binding proteins are known to exist as monomers, dimers, or oligomers depending on their amino acid composition and function (Inman et al. 2001). Most members of the S100 and polcalcin family are highly acidic (Schafer and Heizmann 1996; Niederberger et al. 1999). In contrast, the isoelectric point (pI) of Calsensin is close to physiological pH and hence could be modulated by small changes in the pH. The anti-parallel packing of the helices in Calsensin is comparable to the open conformation of the N-terminal domain of Ca²⁺-bound CaM (Nelson and Chazin 1998a) and monomer of S100 proteins (Potts et al. 1996; Vallely et al. 2002). The packing in polcalcins Bet v 4 and Phl p7 is slightly different due to the extra Z-helix (Verdina et al. 2002; Neudecker et al. 2004).

Backbone dynamics of Calsensin
The global correlation time _c of Calsensin was 6.7± 0.1 nsec, which is comparable to that observed for proteins of similar size at 298 K (Theret et al. 2001b). The molecule has a statistically significant prolate rotational diffusion tensor (D_|/D =1.14), which is consistent with other calcium-binding proteins (Malmendal et al. 1999; Inman et al. 2001). All 74 residues with assigned backbone amide resonances had their corresponding ¹⁵N relaxation data fitted to one of five models describing modes of backbone dynamics (Mandel et al. 1995). A majority of the residues (46 out of 74) were satisfied by model 1, 13 by model 2, three by model 3, 11 by model 4 , and one by model 5 using the nomenclature of Mandel et al. (1995). The fitted order parameters (S²) (Fig. 2E) reveal that the regions of high order correlate with the presence of -helical secondary structural elements. The third helix, suggested to be involved in calciuminduced conformational change in most S100 proteins (Smith et al. 1996; Drohat et al. 1998; Donato 2001) was best fitted by the model having a msec timescale exchange term (R_ex) for four out of eight residues (Fig. 2G). This helix also has lower order parameters as compared to the other three helices.

View larger version (27K): [in this window] [in a new window]   Figure 2. Backbone dynamics of Calsensin correlate with the observed structural features. (A) The secondary structural elements of Calsensin are shown corresponding to the residue number. The transverse relaxation time (T1) (B), longitudinal relaxation time (T2) (C), heteronuclear NOE (D), order parameters (S2) (E), internal motions (F), and exchange rates (G) are plotted as a function of residue number. All experiments were carried out at 298 K.

Calcium-dependent conformational change
The EF-hand family of calcium-binding proteins that function as buffer proteins has similar structures in both the apo- and calcium-bound form (Nelson and Chazin 1998b; Yap et al. 1999). In contrast, calcium sensors that mediate signal transduction undergo a significant calcium-dependent conformational change (Nelson and Chazin 1998b; Yap et al. 1999). The mechanism of the calcium-dependent changes for these proteins has been extensively studied using calcium titrations (Aitio et al. 1999) as well as by solving the apo- and calcium-bound structures (Maler et al. 2002). For example, the calcium-induced structural changes for S100B suggest a large conformational change in the orientation of H3 (Drohat et al. 1998). This reorientation in turn alters the structure of the hinge region and second calcium-binding site. In Calsensin, the relaxation data suggest a high degree of flexibility at the second site on a millisecond timescale (Fig. 2G). Hence, the binding of calcium to the first site might enable a conformational change at the second site allowing calcium-binding at this site. The residues in the hinge region and the C-terminal loop of S100 have been shown to be involved in target binding (Bhattacharya et al. 2003). The binding of calcium leads to a conformational change exposing the hydrophobic residues on the surface (Smith et al. 1996), consequently modulating target binding (Ikura et al. 1992; Malmendal et al. 1999). The hinge region and C-terminal helices of Calsensin consist mostly of hydrophobic residues. This suggests that the calcium-induced structural changes could expose these hydrophobic residues on the molecular surface thereby allowing interaction with target proteins.

Conclusions
Calsensin is a member of the two EF-hand calciumbinding protein family that includes the S100 and polcalcin families. Molecules like Calsensin that are expressed selectively in certain neurons are candidates to function as signal transducers during axon fasciculation and growth cone guidance (Briggs et al. 1995). We have used multidimensional NMR to solve the structure of calcium-bound Calsensin. The structure of Calsensin reveals an anti-parallel stacking of the two helices of each EF-hand. The relatively higher disorder of H3 in the solution structure as compared to other helices is due to the presence of millisecond-timescale conformational exchange. The observed flexibility of H3 could be attributed to chemical exchange between calcium-loaded and free states and/or closed and open conformations. This indicates that the third helix is important for the calcium-induced conformational changes and that it may be implicated in target protein interactions.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Protein expression and purification
The full-length Calsensin ORF (Briggs et al. 1995) was PCR amplified and cloned into the pGEX4T3 vector (Amersham Biosciences) to generate the construct pGEX4T3-Cal. For protein purification Escherichia coli strain BL21 (DE3) was transformed with pGEX4T3-Cal and the cultures grown in 2XYT medium supplemented with 1 mM CaCl₂ or modified M9 medium supplemented with 1 mM CaCl₂ and 100 mg/mL ampicillin. For unlabeled protein expression in 2XYT medium, the cultures grown at 37°C were induced with 0.1 M isopropyl -D-thiogalactopyranoside (IPTG) when O.D.₆₀₀ reached 0.5. For ¹⁵N-single-labeled or ¹⁵N- and ¹³C-doublelabeled protein preparations, the modified M9 minimal media contained ¹⁵N-enriched ammonium chloride (1 g/L [Cambridge Isotope Laboratories]) and/or ¹³C-enriched glucose (2 g/L [Cambridge Isotope Laboratories]) as the sole nitrogen and carbon source, respectively. Cells were grown to O.D.₆₀₀ of 0.6 at 37°C, transferred to 30°C, and induced with 1 mM IPTG when O.D.₆₀₀ reached 0.9. The cells were harvested by centrifugation, 6 h and 12 h post-induction for unlabeled and labeled samples, respectively. The cell pellets were resuspended in 50 mL of 50 mM sodium phosphate buffer (75 mM NaCl, 2 mM DTT and 0.02% NaN₃ [pH 6.0]) per liter of culture with lysozyme added to a final concentration of 1 mg/mL. After freezing at –80°C overnight (Brazin et al. 2000) the cells were disrupted upon thawing and protease inhibitor (1 mM PMSF) and DNase I (500 mL of 1 mg/mL stock) were added. The cell extracts were clarified by centrifugation and the supernatant loaded on a gluthathione-agarose (Sigma) column. The GST-fusion proteins were eluted with 5 mM reduced glutathione in 50 mM sodium phosphate buffer, concentrated with a Millipore stirred ultrafiltration cell, and separated on a size-exclusion column (Sephacryl S-100 HR, Amersham Pharmacia Biotech) equilibrated with 50 mM sodium phosphate buffer. Fractions containing the fusion proteins were pooled, the NaCl concentration increased to 150 mM, and the GST-tag cleaved off with thrombin by incubation at room temperature for 12–16 h. The GST-tag was subsequently removed from the recombinant Calsensin protein using a glutathione-agarose column. The Calsensin protein was further purified by gelfiltration (Sephacryl S-100 HR). The collected fractions were analyzed by SDS-PAGE for purity, pooled, and concentrated to 1–2 mM for NMR experiments. The final NMR samples contained 10% ²H₂O.

NMR spectroscopy
The NMR samples were prepared in 50 mM sodium phosphate buffer (pH 6.0) containing 75 mM NaCl, 2 mM DTT and 0.02% NaN₃. All NMR data were acquired at 298 K on a Bruker DRX500 spectrometer operating at ¹H frequency of 499.867 MHz. A 5-mm triple-resonance (¹H/¹⁵N/¹³C) probe with XYZ field gradients was used for all experiments. A gradient-enhanced HSQC experiment with minimal water saturation (Mori et al. 1995) was used for all ¹H-¹⁵N correlation experiments. 3D ¹⁵N-edited TOCSY, ¹⁵N-edited NOESY (Talluri and Wagner 1996) and ¹⁵N-HMQC-NOESY-HMQC (Andersson et al. 1998) spectra were collected for ¹⁵N-labeled Calsensin sample using mixing times of 80 msec for TOCSY and 125 msec for NOESY experiments. For ¹⁵N/¹³C doubledlabeled samples 3D CBCA(CO)NH, HNCACB (Muhandiram and Kay 1994), CCONH (Muhandiram and Kay 1994), HCCH-TOCSY (Kay et al. 1993), and ¹³C-edited NOESY (Muhandiram et al. 1993) spectra were acquired using standard experimental procedures. The backbone coupling constants (³JNH-H) were measured by a HNHA (Kuboniwa et al. 1994) experiment. Additionally, 2D homonuclear ¹H-¹H TOCSY (Fulton et al. 1996), NOESY (Lippens et al. 1995), as well as DQF-COSY (Piantini et al. 1982) data were obtained. Deuterium exchange experiments were performed as described by Roberts (1993). The proton chemical shifts were referenced to DSS (Markley et al. 1998) and ¹⁵N and ¹³C chemical shifts were referenced indirectly. The data were processed on a Linux workstation using NMRPIPE software package (Delaglio et al. 1995) and assignments were carried out using NMRView (Johnson and Blevins 1994).

Structure calculation
The NOE and distance restraints were generated using resonance assignments from 2D and 3D data sets analyzed with NMRView. Additionally, the deuterium exchange as well as the backbone dynamics data were interpreted using NMRView. The peak volumes obtained from NOEs were classified as strong, medium, weak, and very weak restraints, corresponding to upper bound interproton distances of 2.8, 3.4, 4.3, and 5.0–6.0 Å. Pseudo-atom corrections were added for methylene and methyl protons (Wuthrich 1986). The interproton distances and backbone torsion angle constraints served as input for structure calculations using distance geometry and simulated annealing with CNS version 1.1 (Brunger et al. 1998). Hydrogen bond constraints of rNH-O 1.5–2.8 Å and rN-O=2.4–3.5 Å were introduced during structure calculations based on ²H₂O exchange data, and in the regions of secondary structure having characteristic NOEs. The refinement of 200 structures yielded several structures (>150) with no distance violations greater than 0.4 Å and no dihedral angle violations greater than 5°. The final 20 structures were selected on the basis of lowest total energies and having minimal restraint violations. The statistics of the 20 lowest energy structures are represented in Table 1 and the coordinates have been deposited in the Protein Data Bank (accession number 6519). All the structures were visualized and rendered using MOLMOL (Koradi et al. 1996). The complete resonance assignments have been deposited into BioMagRes Bank as accession codes 1YX7 and 1YX8.

Relaxation data analysis
Measurement of ¹⁵N longitudinal relaxation rates R₁, transverse relaxation rates R₂ and {¹H}-¹⁵N NOE were obtained at 11.7 T and 298 K as previously described (Farrow et al. 1994 [refer to Fig. 10 for recycle delays]). The auto-relaxation rate constants R₁ and R₂ were calculated by nonlinear optimization using the rate analysis tool in NMRView. The heteronuclear NOEs were obtained as a ratio of HSQC cross-peak intensities measured with and without steady-state saturation of proton magnetization (Farrow et al. 1994). The global correlation time _c was calculated using 53 residues after excluding residues for which the resonance frequencies overlap, or those that undergo large-scale internal motions and/or conformational exchange (Tjandra et al. 1995). The rotational diffusion tensor was determined for the energy minimized average structure and the ¹⁵N relaxation parameters using TENSOR2 (Dosset et al. 2000). The data were analyzed using extended model-free formalism with the statistical model selection of Mandel et al. (1995) as implemented in TENSOR2.

	Acknowledgments

 
This work was supported by NIH grant NS28857. We thank Dr. Monica Sundd for valuable discussions during the preparation of this manuscript. We also appreciate the technical help provided by Jayandran Palaniappan for structure calculations.

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