Molecular dynamics of the neuronal EF-hand Ca2+-sensor Caldendrin

Abstract

Caldendrin, L- and S-CaBP1 are CaM-like Ca2+-sensors with different N-termini that arise from alternative splicing of the Caldendrin/CaBP1 gene and that appear to play an important role in neuronal Ca2+-signaling. In this paper we show that Caldendrin is abundantly present in brain while the shorter splice isoforms L- and S-CaBP1 are not detectable at the protein level. Caldendrin binds both Ca2+ and Mg2+ with a global Kd in the low µM range. Interestingly, the Mg2+-binding affinity is clearly higher than in S-CaBP1, suggesting that the extended N-terminus might influence Mg2+-binding of the first EF-hand. Further evidence for intra- and intermolecular interactions of Caldendrin came from gel-filtration, surface plasmon resonance, dynamic light scattering and FRET assays. Surprisingly, Caldendrin exhibits very little change in surface hydrophobicity and secondary as well as tertiary structure upon Ca2+-binding to Mg2+-saturated protein. Complex inter- and intramolecular interactions that are regulated by Ca2+-binding, high Mg2+- and low Ca2+-binding affinity, a rigid first EF-hand domain and little conformational change upon titration with Ca2+ of Mg2+-liganted protein suggest different modes of binding to target interactions as compared to classical neuronal Ca2+-sensors.

Figure 1. Expression anaylsis of Caldendrin, L- and S-CaBP1.

(A) Immunoblot analysis reveals that bacterially expressed untagged Caldendrin migrates at 33 kDa like the smaller Caldendrin isoform in cortex and hippocampus of rat brain. Bacterially produced myristoylated L- and S-CaBP1 migrate at 25 kDa and 18 kDa respectively. Immunoreactivity is detected by anti-Caldendrin/CaBP1 rabbit antibody, directed against the common C-terminus of all three isoforms. 20 µg of brain samples are compared to ≈ 10 ng of purified proteins. The western blot shows Caldendin expression in different regions of rat brain (B) and in different rat organs (C). Caldendrin is detected by anti-Caldendrin/CaBP1 rabbit antibody (rb). Equal loading in all lanes was ensured by measuring the total protein concentraion (20 µg/lane) and verified with an anti-actin mouse antibody (ms). Note that consistant with previous reports the actin band is amost absent in heart tissue due to differnential expression of this marker.

Figure 2. Extrinsic fluorescence ANS (1-Anilino-8-Naphthalin Sulfonate) fluorescence spectroscopy of Caldendrin and L- and S-CaBP1.

(A) Mg²⁺ and Ca²⁺ titration to Mg²⁺ bound apo Caldendrin shows no change in the fluorescence intensity. (B) Titration of Mg²⁺ and Ca²⁺ titration to Mg²⁺ bound L-CaBP1 shows no change in the fluorescence intensity. (C) Effect of Mg²⁺ and Ca²⁺ on apo S-CaBP1. Titration with Mg²⁺ results in a significant decrease in the fluorescence intensity, whereas Ca²⁺ titration to Mg²⁺ bound S-CaBP1 reversed this effect. (D) Titration with Mg²⁺ affects tryptophane fluorescence intensity in EF1W mutant Caldendrin, (E) EF3W mutant Caldendrin exhibits large changes in tryptophane fluorescence intensity following Mg²⁺ and Ca²⁺ titration. For all titrations a protein concentration of 1 µM was used. The Mg²⁺ concentration was 0.5 mM and the Ca²⁺ concentration was 50 µM.

Figure 3. Far and Near UV- CD spectroscopy.

(A) Far UV- CD spectra of the apo, Mg²⁺-bound and Mg²⁺+Ca²⁺ bound common C-terminus of Caldendrin/CaBP1 shows no conformational change in secondary structure. (B) Near UV- CD spectra of the apo, Mg²⁺-bound and Mg²⁺+Ca²⁺ bound common C-terminus of Caldendrin/CaBP1 exhibit significant change in tertiary structure. (C) Far UV- CD spectra of the apo-, Mg²⁺-bound and Mg²⁺+Ca²⁺-bound full length Caldendrin. Mg²⁺ binding to apo-Caldendrin causes a mild change in the secondary structure of the protein. Ca²⁺ binding to Mg²⁺- bound Caldendrin has no detectable effect on the secondary structure of the protein. (D) Near UV- CD spectra of apo-, Mg²⁺-bound and Ca²⁺-bound Mg²⁺+Ca²⁺-bound full length Caldendrin. Mg²⁺ binding to apo-CDD causes a significant change in the tertiary structure of the protein, whereas Ca²⁺ binding to Mg²⁺- bound Caldendrin has no effect. (E) Ca²⁺-titration of the apo-protein had a clear effect on the structure. Representative spectra for each condition are shown.

Figure 4. Equilibrium chemical unfolding.

Equilibrium chemical unfolding of full length Caldendrin with guanidinium hydrochloride (GdmCl) in the absence of any ligand (Apo-CDDfl), in presence of 5 mM MgCl₂ and 1 mM EGTA (Mg²⁺-CDDfl); and 5 mM MgCl₂ and 1 mM CaCl₂ (Mg²⁺Ca²⁺- CDDfl). The upper lane shows (A) the ellipticity values of plotted against [GdmCl] (black squares) and the fitting obtained (black curve) using a two-state unfolding model. The middle lane (B) shows the quality of model fitting in terms of residuals and the bottom lane (C) shows the normalized fits for fraction folded (FN; red curve) and fraction unfolded (FU; black curve) along with the standard free energy change of unfolding (ΔGU) obtained.

Figure 5. ITC analysis of Caldendrin and the common C-terminus of Caldendrin, L- and S-CaBP1.

(A) Cartoon showing the Caldendrin (CDD) full length (1–298 residues) and Caldendrin Ct (137–298 residues) proteins used for ITC. (B) ITC of Apo-Caldendrin with Mg²⁺. Titration was carried out with 10 mM MgCl₂. Protein concentration was 43 µM. Data were fitted using a sequential binding model. (C) Ca²⁺ titration to Mg²⁺ saturated Caldendrin. Titration was carried out with 10 mM CaCl_2. Data were fitted using a sequential binding model. (D–F) ITC analysis of Mg²⁺, Ca²⁺ and Ca²⁺ titration to the Mg²⁺ bound common C-terminus of Caldendrin/CaBP1 (173 µM). The upper part in each in each panel shows the traces of calorimetric ion titration and the lower panel represents the integrated binding isotherms obtained from various best fit models. This figure is connected to Table 1 which includes the corresponding ITC values.

Figure 6. Surface plasmon resonance analysis.

(A) Untagged full length Caldendrin was immobilized on a CM5 surface and the full-length Caldendrin was also injected as analyte. (B) The N-terminus of Caldendrin was immobilized on a CM5 surface and also injected as analyte in running buffer. (C) The common C-terminus (CDD-Ct) was immobilized on the sensor chip and the N-terminus injected as analyte. (D) The common C-terminus (CDD-Ct) was immobilized on the sensor chip and also injected as the analyte. (A–D) The running buffer always contained 50 mM Tris-Cl and 100 mM KCl with 1 mM Mg²⁺/1 mM EGTA (red) or 1 mM Mg²⁺/500 µM Ca²⁺ (green). Amount of protein in the running buffer was 5, 10, 20, 40, and 80 µg (increasing protein levels correspond to increasing amplitudes). RU: response units.

Figure 7. Dynamic light scattering (DLS) experiments further demonstrate the existence of a Caldendrin homodimer.

(A) The radius of Caldendrin in the presence of Mg²⁺. Polydispersity suggests that Mg²⁺-bound Caldendrin may exhibit conformational heterogeneity or exist as an equilibrium mixture of monomer and dimer species. (B) DLS shows that Caldendrin in the Ca²⁺-bound state preferentially forms a dimer. (C) Table showing the molecular mass of Caldendrin, polydispersity in the presence of Mg²⁺ (1 mM Mg^2+/1 mM EGTA) or Mg²⁺+Ca²⁺ (1 mM Mg²⁺/50 µM Ca²⁺). Experiments were repeated with three different purifications and from each puriifcation samples were measured six to seven times.