Interactions of the Anti-Psychotic Drug Trifluoperazine with Calmodulin

Abstract

Calmodulin (CaM) is a Ca2+-sensing protein essential to eukaryotic signal transduction pathways. It has two homologous domains (N and C), each binding two Ca2+ ions. The anti-psychotic drug trifluoperazine (TFP; Stelazine) is a CaM antagonist known to bind hydrophobic clefts of CaM that are exposed upon Ca2+ binding. Equilibrium Ca2+ titrations monitored by changes in steady-state fluorescence of intrinsic Phe and Tyr residues were used to evaluate the effect of TFP on the Ca2+ affinity of full length CaM (CaM1–148), N-domain (CaM1–80) and C-domain (CaM76–148) over a range of TFP:CaM ratios. Low levels of TFP (1:1, 2:1 ratios) decreased the Ca2+ affinity of CaM. TFP had the greatest effect on Ca2+ binding to sites III and IV, in the C-domain of CaM1–148, but affected both domains. At an 8:1 ratio of TFP:CaM, the effect reversed and the Ca2+ affinity of CaM increased. 1H-15N-HSQC NMR showed that resonances assigned to apo and Ca2+-saturated C-domain were the most perturbed during TFP titration, while a smaller subset of N-domain resonances were affected. The stoichiometry of TFP binding to apo-CaM1–148 was determined to be 2:1, and 4:1 for (Ca2+)4-CaM. Crystallographic structures of TFP bound to (Ca2+)4-CaM1–148 indicate two possible orientations of TFP when bound in 1:1 vs 2:1 and 4:1 TFP:CaM ratios. A new structure of a (Ca2+)2-CaM76–148 -TFP complex showed the trifluoromethyl group of TFP in both positions seen previously; distinct conformation of Met 144 correlated with orientation of TFP. NMR of apo-CaM76–148 will be used to determine whether apo CaM-TFP complex adopts the semi-open conformation of apo CaM bound to a myosin peptide.