Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin

Abstract

Trifluoperazine (TFP; Stelazine) is an antagonist of calmodulin (CaM), an essential regulator of calcium-dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca(2+))(4)-CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the "open" conformation seen in CaM-kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca(2+))(4)-CaM and explore differential effects on the N- and C-domains of CaM, stoichiometric TFP titrations of CaM were monitored by (15)N-HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca(2+))(4)-CaM. In both cases, the preferred site was in the C-domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP-binding sites in apo CaM appeared distinct from those in (Ca(2+))(4)-CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ-motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N-domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the "closed," "semi-open," and "open" domains of CaM. In physiological processes, apo CaM, as well as (Ca(2+))(4)-CaM, needs to be considered a potential target of drug action.

Figure 1. Structural Background of CaM-Target Interactions

A: Superposition of models of the solution structures of CaM (1CFC.pdb) that have been determined by NMR. Left: Alignment minimized the difference between models with respect to the N-domain (residues 1-75 in blue), illustrating the flexibility of the interdomain linker (resides 76-80 in black) and range of relative positions adopted by the C-domain (residues 81-148 in red). A single model is highlighted to reveal the tertiary structures of the apo N- and C-domains (each is a 4-helix bundle). Middle: (Ca²⁺)₄-CaM structure determined crystallographically (1CLL.PDB); backbone colored as in panel A. Ca²⁺ ions (yellow) are bound at sites I and II in the N-domain, and at sites III and IV in the C-domain. Right: Chemical structure of the antipsyschotic drug Trifluoperazine (TFP; green), with sulfur atom in yellow and fluorine atoms in light blue. Figure created with Pymol™. B: Superposition of 3 Tertiary Structures of the C-domain of CaM. Examples of the “closed” (1CFC.pdb–orange), “semi-open” (2IX7.pdb–green) and “open” (1CDM.pdb–aqua and 1CLL.pdb–magenta) conformations of the C-domain of CaM are aligned according to the positions of the F and G (second and third) helices of the domain. Structures of the corresponding full-length CaM is shown below. Figure created with Pymol™. C: Structures of (Ca²⁺)₄-CaM bound to TFP. Individual panels show crystallographically derived structures of TFP:(Ca²⁺)₄-CaM complexes, with drug:protein ratios of 1:1 (1CTR.pdb), 2:1 (1A29.pdf), and 4:1 (1LIN.pdb), as well as a structural superposition of these three structures, with the TFP-binding sites labeled A (green), B (magenta), C (brown) and D (orange). TFP-binding sites A and B are located in the C-domain (backbone red), site C bridges the two domains, and site D is located in the N-domain (backbone blue). Figure created with Pymol™.

Figure 2. ¹⁵N-HSQC-monitored TFP titration of uniformly ¹⁵N-labeled apo PCaM

A: Comparison of subset of ¹⁵N-HSQC spectra for apo PCaM (black) and TFP-saturated apo PCaM (red); arrows indicate change in resonance positions over the course of the TFP titration. B: Normalized TFP-induced chemical shifts of individual representative residues of apo CaM. C: Bar graph of net chemical shift per residue caused by TFP saturation of CaM. D: Location of each apo CaM residue whose chemical shift was perturbed > 0.05 ppm by TFP saturation (white spheres); backbone modeled as that of apo CaM (1DMO.pdb). Solution conditions: 10% D₂O, 10 mM imidazole, 100 mM KCl, 50 μM EDTA, 5mM, pH 6.5 at 22°C. Figure created with Pymol™.

Figure 3. ¹⁵N-HSQC-monitored TFP titration of uniformly ¹⁵N-labeled (Ca²⁺)₄-CaM

A: Comparison of (Ca²⁺)₄-CaM (black) and TFP-saturated (Ca²⁺)₄-CaM (red) ¹⁵N-HSQC spectra, arrows indicate change in resonance positions over the course of the TFP titration. B: Normalized TFP-induced chemical shifts of individual representative residues of (Ca²⁺)₄-CaM. C: Bar graph of net chemical shift per residue caused by TFP saturation of CaM. D: Location of each (Ca²⁺)₄-CaM residue whose chemical shift was perturbed > 0.05 ppm by TFP saturation (white spheres); backbone modeled according to the structure of TFP bound to (Ca²⁺)₄-CaM at a 4:1 ratio (1LIN.pdb). Solution conditions: 10% D₂O, 10 mM imidazole, 100 mM KCl, 50 μM EDTA, 5mM CaCl₂, pH 6.5 at 22°C. Figure created with Pymol™.

Figure 4. Multiple chemical environments observed upon TFP titration of (Ca²⁺)₄-CaM

A: ¹⁵N-HSQC spectrum of uniformly ¹⁵N labeled (Ca²⁺)₄-CaM titrated with TFP, where arrows represent the movement of each resonance from its initial position. B: Schematic diagram of quantitative criterion for classification of biphasic chemical shift. C: Locations of select residues that underwent a biphasic response upon TFP addition, mapped onto the structure of TFP bound to (Ca²⁺)₄-CaM at a 4:1 ratio (1LIN.pdb). D: Normalized chemical shift plots for select individual residues deemed to undergo a biphasic response upon TFP titration of (Ca²⁺)₄-CaM. Solution conditions: 10% D₂O, 10 mM imidazole, 100 mM KCl, 50 μM EDTA, 5mM CaCl₂, pH 6.5 at 22°C. Figure created with Pymol™.

Figure 5. Effect of TFP on Calcium Binding to CaM_1-148

Equilibrium calcium titrations of CaM (6 μM) were conducted in the presence of 0 (blue), 6 (green, 1:1), 12 (red, 2:1), 18 (black, 3:1), 24 (cyan, 4:1), or 48 μM (purple, 8:1 TFP:CaM) TFP, and were monitored using the intrinsic fluorescence of CaM. A: phenylalanine fluorescence (250 nm_ex and 280 nm_em). B: tyrosine fluorescence (277 nm_ex and 320 nm_em). In B, for 3:1, 4:1 and 8:1 TFP:CaM, the raw signal decreased; it is shown inverted to facilitate comparisons. Solid curves were simulated according to Eq. 3 and free energies in Table 1; bar graph insets represent ΔΔG₂ values in Table 1. Solution conditions: 50 mM HEPES, 100 mM KCl, 5 mM KCl, 0.05 mM EGTA, 1 mM MgCl₂, and 6 nM Oregon Green (pH 7.4) at 22°C.

Figure 6. Effect of TFP on Calcium Binding to CaM_1-80 and CaM_76-148

Equilibrium calcium titrations of CaM (6 μM) were conducted in the presence of 0 (blue), 6 (green, 1:1), 12 (red, 2:1), 18 (black, 3:1), 24 (cyan, 4:1), or 48 μM (purple, 8:1 TFP:CaM) TFP, and were monitored using the intrinsic fluorescence of CaM. A: phenylalanine fluorescence (250 nm_ex and 280 nm_em). B: tyrosine fluorescence (277 nm_ex and 320 nm_em). In B, for 2:1 and 3:1 TFP:CaM, only the first transition is show. In B, for 4:1 and 8:1 TFP:CaM, the raw signal decreased; it is shown inverted to facilitate comparisons. Solid curves were simulated according to Eq. 3 and free energies in Table 1; bar graph insets represent ΔΔG₂ values in Table 1. Inset in Figure 6B are calcium titration of CaM_76-148 at 12 μM (red, 2:1 TFP:CaM) and 18 μM (black, 3:1 TFP:CaM) monitored using the intrinsic tyrosine fluorescence of CaM (277 nm_ex and 320 nm_em). Evidence for multiple species, and piecewise analysis described in Results. Solid curves for calcium-dependent increase in fluorescent intensity were simulated according to Eq. 3 and free energies in Table 1; dashed curves correspond to decrease in fluorescence intensity.Solution conditions were 50 mM HEPES, 100 mM KCl, 5 mM KCl, 0.05 mM EGTA, 1 mM MgCl₂, and 6 nM Oregon Green (pH 7.4) at 22°C.

Figure 7. TFP-saturated apo and (Ca²⁺)₄-CaM_1-148

A. Overlay of ¹⁵N-HSQC spectra of apo CaM (black, 2 TFP:CaM) and (Ca²⁺)₄-CaM (red, 4 TFP:CaM). Few peaks overlay, indicating significantly different chemical environments for backbone amides in the structures of apo and (Ca²⁺)₄-CaM saturated by TFP. B. Interdomain interactions in (Ca²⁺)₄-CaM_1-148 mediated by TFP. Based on the crystal structure with 4:1 TFP:(Ca²⁺)₄-CaM (1LIN.pdb), the TFP molecule shown in ball-and-stick (brown, with fluorine, sulphur, and nitrogen atoms in green, yellow, and blue respectively) interacts with residues in both the calcium-saturated N-domain (blue) and C-domain (red). Those within 4 Å were residues 8, 11, 72, 92, 144, 145, TFP 1, and TFP 2. (Ca²⁺)₄-CaM backbone (gray), 4 calcium ions (yellow spheres), and three other TFP (gray sticks) are shown. Figure created with Pymol™.

Figure 8. Docking and Models of TFP binding to CaM C-domain

A. TFP Docking to alternative tertiary conformations of apo CaM. Ribbon diagrams of C-domain fragments (residues 82 to 146) represent the “closed” (1DMO.pdb), “semi-open” (2IX7.pdb) and “open” (2HQW, 1LIN.pdb) conformations. Calcium was removed from 2HQW and 1LIN. AutoDock Vina 1.0.3 predicted positions of TFP binding; 20 models having lowest free energy are shown as sticks. The single sulfur atom of each TFP is shown as a sphere; green corresponds to the most favorable free energy of binding; white is the least favorable. Color thermometer below each set of models indicates the range of energies predicted. The TFP molecule observed at site A of 1LIN.pdb is shown in magenta. Residues in calcium-binding sites are yellow; arrows are included only to orient the viewer to chain direction. B. Model of conformational transition of apo C-domain in equilibrium between a “closed” and “semi-open” conformation. Binding of TFP to the blue patches accessible in the “semi-open” conformation is energetically more favorable than binding to “closed” form. TFP binding to the blue occludes hydrophobic patches show in purple of the apo C-domain that are otherwise exposed to solvent. An “open” conformation is adopted upon calcium binding, whether alone or also bound to a drug or protein target exposing hydrophobic patches shown in purple.