Opposing orientations of the anti-psychotic drug trifluoperazine selected by alternate conformations of M144 in calmodulin

Abstract

The anti-psychotic drug trifluoperazine (TFP) is an antagonist observed to bind to calcium-saturated calmodulin ((Ca(2+) )4 -CaM) at ratios of 1:1 (1CTR), 2:1 (1A29), and 4:1 (1LIN). Each structure contains one TFP bound in the hydrophobic cleft of the C-domain of CaM. However, the orientation of the trifluoromethyl (CF3 ) moiety differs among them: it is buried in the C-domain cleft of 1A29 and 1LIN, but protrudes from 1CTR. We report a 2.0 Å resolution crystallographic structure (4RJD) of TFP bound to the (Ca(2+) )-saturated C-domain of CaM (CaMC ). The asymmetric unit contains two molecules of (Ca(2+) )2 -CaMC . Chain backbones were nearly identical, but the orientation of TFP in the cleft of Chain A matched 1A29/1LIN, while TFP bound to Chain B matched 1CTR. This was accommodated by a flip of the M144 sidechain and small changes in sidechains of M109 and M145. Docking simulations suggested that the rotamer conformation of M144 determined the orientation of TFP within the cleft of (Ca(2+) )2 -CaMC . Chains A and B show that the open cleft of (Ca(2+) )2 -CaMC is promiscuous in accepting TFP in reversed directions under the same crystallization conditions. Observing multiple orientations of an antagonist bound to a single protein highlights the challenge of designing highly specific pharmaceuticals, and may have importance for QSAR of other CF3 -containing drugs such as fluoxetine (anti-depressant) or efavirenz (reverse transcriptase inhibitor). This study emphasizes that a single structure of a complex represents an energetically accessible state, but does not necessarily show the full range of energetically equivalent states.

Keywords: CF3; QSAR; TFP; alternate conformers; drug specificity; energetics; molecular recognition; pharmaceuticals; promiscuous binding; thermodynamics; trifluoromethyl.

Figure 1

A: Structures of TFP bound at a common C-domain site of (Ca²⁺)₄-CaM_1–148 and chemical structure of TFP. Superimposed structures of the α-carbon backbone atoms of TFP bound to the C-domain (red) of (Ca²⁺)₄-CaM_1–148 (N-domain not shown), with calcium ions (yellow spheres), and TFP (sticks) colored green, purple and brown corresponding to structures of TFP bound to (Ca²⁺)₄-CaM_1–148 at ratios of 1:1 (1CTR.pdb), 2:1 (1A29.pdb), and 4:1 (1LIN.pdb), respectively. Inset box shows the chemical structure of TFP highlighting the position of the CF₃ moiety. B: (Ca²⁺)₄-CaM residues involved in TFP binding at a common site in (Ca²⁺)₄-CaM_1–148. Position of sidechain atoms of (Ca²⁺)₄-CaM residues within 4Å of the common TFP binding site in TFP/(Ca²⁺)₄-CaM_1–148 complexes superimposed on the basis of their α-carbon backbone atoms are shown in light green (1:1), light purple (2:1), and light brown (4:1) sticks. TFP is shown in transparent sticks where the position of the CF₃ group in the 1:1, 2:1, and 4:1 structures has been highlighted to depict the 180° flip in TFP orientation. C: Structure of TFP bound (Ca²⁺)₂-CaM_C. On top, chain A (red) and chain B (orange) are shown as ribbons inside of their transparent molecular surfaces; calcium ions (yellow spheres), and TFP (sticks) are highlighted. On bottom, the α-carbon backbone atoms of chains A and B are superimposed to illustrate the similarity in their backbone conformations. D: Location and 2Fo-Fc electron density map of TFP molecules bound to (Ca²⁺)₂-CaM_C at 1σ contour level. On top, (Ca²⁺)₂-CaM_C is shown in light gray ribbons, calcium ions are shown as light yellow spheres, and TFP (green, magenta, light blue, and pink) arrayed between chain A and B are shown as sticks inside of spheres displaying the van der Waals radii of each TFP atom. Shown below are TFP molecules 1 through 4 fit into their corresponding 1σ 2Fo-Fc electron density maps.

Figure 2

A: Surface representation of (Ca²⁺)₂-CaM_C with bound TFP. Area representing residues within 4Å of either TFP-3 (green) bound to chain A (red) or TFP-1 (magenta) bound to chain B (orange) are colored black. The fluorine atoms of the CF₃ groups of TFP are shown as spheres to highlight the 180° rotation of TFP bound to chain A vs. chain B. B: Comparison of TFP bound within the hydrophobic pocket of (Ca²⁺)₂-CaM_C and (Ca²⁺)₄-CaM. On left, α-carbon backbone atoms of chain A (red) were superimposed with the C-domain of the 1:1 TFP/(Ca²⁺)₄-CaM complex in 1CTR (cyan). On right, α-carbon backbone atoms of chain B (orange) were superimposed with the C-domain of the 4:1 TFP/(Ca²⁺)₄-CaM complex in 1LIN (light green). TFP are shown as green (chain A), magenta (chain B), yellow (1:1), and cyan (4:1) sticks. Calcium ions are represented as yellow spheres. C: Comparison of (Ca²⁺)₂-CaM_76–148 TFP binding sites in clefts of chain A and B. The α-carbon backbone atoms of chain A (red) were superimposed with those of chain B (orange). Sidechain atoms within 4Å of the corresponding TFP binding site are shown as sticks, with M144 colored green (chain A) and magenta (chain B). Calcium ions are represented as yellow spheres. D: A morph between chain A (blue) and B (red) generated by the Yale Morph Server and illustrated using PyMOL. Calcium ions shown as yellow spheres having 60% VDW radius. E: The local protein contacts potential of chain A was generated using PyMOL. F: Comparison of sidechain rotamer conformations adopted by M144 in (Ca²⁺)₂-CaM_C chain A (top) and B (bottom) in relation to TFP 1 (green) and TFP 4 (magenta); van der Waals radii of each atom are represented as spheres. G: Docking simulations of TFP binding to (Ca²⁺)₂-CaM_76–148 using Autodock Vina 32. Chains A (red) and B (orange) of (Ca²⁺)₂-CaM_C are shown as ribbons. The experimentally observed positions of TFP in each chain are shown in green (chain A) and magenta (chain B). The 10 lowest energy positions of TFP are shown in transparent sticks; fluorine atoms of the CF₃ group are blue spheres.