Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium

Abstract

Background: Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR.

Results: We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified.

Conclusion: Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Keywords: CDZ; CaM; Calmidazolium; Calmodulin; Calmodulin antagonist; Protein dynamics; Structure.

Fig. 1

SAXS patterns and DENSS analysis of holo-CaM in the absence and in the presence of CDZ. A Scattering patterns of holo-CaM (blue trace) and holo-CaM:CDZ complex (red trace). B Distance distribution function P(r) of holo-CaM in the absence and in the presence of CDZ using GNOM, same color code as in panel A. C Dimensionless Kratky representation of holo-CaM in the absence and in the presence of CDZ, same color code as in panel A. Dash lines crossing x-axis (√3) and y-axis (1.104) indicate the characteristic peak for globular proteins. D, E DENSS models of holo-CaM and holo-CaM:CDZ, respectively

Fig. 2

Structural models of holo-CaM using EOM and structures of holo-CaM:CDZ determined by X-ray crystallography. A Final ensemble of SAXS-derived conformations of holo-CaM using EOM. The four structural models (SASBDB ID: SASDNX3) are superimposed by aligning their N-lobe alpha carbons (residues 6 to 66) using Pymol. One structural model is shown in rainbow, the three others are in grey. B Comparison of experimental data (grey dots) to the calculated scattering pattern (blue curve) of the final EOM ensemble. C Fitting of the four EOM structural models of holo-CaM to the SAXS-derived DENSS volume. D X-ray structures of holo-CaM in complex with one (red, PDB ID: 7PSZ) and two (green, PDB ID: 7PU9) CDZ molecules. E Superimposition of the CDZ molecules of both structures showing the rotation of the chlorophenyl moieties. F Top: Fitting of the calculated scattering patterns of the two crystallographic structures of holo-CaM:CDZ obtained using Crysol to the experimental SAXS pattern recorded with 333 μM of CaM and 1050 μM of CDZ. The χ² are 1.4 and 9.3 for the 1:1 (red) and 1:2 (green) holo-CaM:CDZ complexes, respectively. Bottom: Distribution of reduced residuals corresponding to the two fits presented above. G Fitting of the two crystallographic structures of holo-CaM:CDZ to the SAXS-derived DENSS volume (SASBDB ID: SASDNY3)

Fig. 3

Effects of CDZ binding on the deuterium uptake profile of holo-CaM. A, B Relative fractional uptake plots of holo-CaM measured in the presence and in the absence of 20 μM CDZ. Each dot corresponds to the average uptake value measured in three independent replicates. C The effects of CDZ binding on holo-CaM are visualized on the fractional uptake difference plot. Negative values indicate a reduction in solvent accessibility induced by CDZ binding. D Cartoon representation of holo-CaM showing the average differences in “fractional uptake differences” between the CDZ-bound and free holo-CaM states. The fractional uptake differences ([ΔDeuteration] in %) measured between the CDZ-bound and free states were extracted for each peptide at each labelling time point, averaged, and plotted on the crystal structure of CaM:CDZ_A (PDB ID: 7PSZ). CDZ-A is colored in green. The average ΔDeuteration values [Average (ΔDeuteration)] are colored from blue (no variation) to red (major reductions in uptake)

Fig. 4

CDZ binding monitored by CaM ¹H-¹⁵N chemical shift perturbation (CSP). A (left) ¹H-¹⁵N SOFAST full fingerprint spectra (recorded at 37 °C) of holo-CaM alone (mauve) and in the presence of 1.0 equivalent of CDZ (green). (right) Zoom on two selected regions of the fingerprint spectra. The spectrum of holo-CaM in the presence of 0.5 equivalents (cyan) is also displayed. The assignments of free holo-CaM are shown and the dotted lines indicate the corresponding signal in the 1:1 holo-CaM:CDZ sample. B CSP values of 1 CDZ equivalent added to holo-CaM as a function of the residue number. The secondary structure (helix=cylinder, strand=arrow), calcium binding loops (spheres and square brackets), and linker region (grey line) are schematized. The CSP values between the 1:1 and 1:2 holo-CaM:CDZ complexes are displayed on the top of the panel, respecting the same scale. The positions of contacting residues in the X-ray 1:1 and 1:2 holo-CaM:CDZ complex structures are represented by wine and maroon rectangles, respectively. Grey lines represent the CSP values chosen as thresholds for very strong and strong CSPs. C Residues with very strongly perturbed (red, CSP ≥ 0.14) and strongly perturbed (orange, 0.07 ≤ CSP < 0.14) amide resonances are highlighted on the cartoon representation of the 1:1 holo-CaM:CDZ x-ray complex structure. CDZ is shown as blue sticks and Ca²⁺ ions as green spheres. The side chains of the CaM residues in close contact with CDZ as defined by Ligplot+ are highlighted as spheres if assigned (19, 36, 39, 54, 63, 71, 84) or as sticks (51, 72, 76, 77, and 145) if not observed by NMR. Amide resonances of the following residues were not assigned in the holo-CaM:CDZ complexes: 8, 12, 14, 16, 38, 51–52, 72, 75–79, 82–83, 88, 92, 106–107, 112, 114, 124, 126–127, 129–130, 139, 143-146

Fig. 5

Decrease of backbone amide internal motion amplitudes in the nanosecond-picosecond time scale of holo-CaM upon binding to CDZ. The difference of the heteronuclear ¹H-¹⁵N nOe of CDZ bound and free holo-CaM is color-coded on the cartoon representation of the holo-CaM:CDZ 1:1 X-ray structure. Red indicates very high positive nOe differences (≥ 0.18) and orange high nOe differences (between 0.10 and 0.16), denoting a decrease in the amplitude of fast internal motions (ns-ps) in the complex. CDZ is shown as blue sticks, Ca²⁺ ions are displayed as green spheres and the linker residues are shown in grey. The side chains of the CaM residues in close contact with CDZ as defined by Ligplot+ are highlighted as spheres if assigned (19, 36, 39, 54, 63, 71, 84) or as sticks (51, 72, 76, 77, and 145) if not observed by NMR

Fig. 6

Effect of CDZ binding on calmodulin. CDZ binding dramatically affects the conformation of CaM, which collapses from a dumbbell-shaped conformation into a compact globular structure in which the CaM lobes wrap around CDZ. A single CDZ molecule is enough to induce dynamics and structural changes of CaM. The N-lobe and C-lobe of holo-CaM are depicted in blue and red, respectively