Effect of Trifluoperazine on Ca2+ -Bound Calmodulin binding to Fas Death Domain for DISC Formation

Abstract

Fas death receptor-activated signaling pathway is one important regulating mechanism of apoptosis in a variety of cells. The formation of the death inducing signaling complex (DISC) is a critical step for Fas-mediated signaling of apoptosis. Recent experimental studies showed that calmodulin (CaM) binds to Fas and regulates Fas-mediated DISC formation and the binding of CaM to Fas is inhibited by CaM antagonist, trifluoperazine (TFP). However, the exact molecular mechanisms for the effect of TFP on Fas-mediated DISC formation are still unknown. Knowledge about these is important for identifying new drug candidate to regulate Fas-mediated signaling pathway for apoptosis. In this study, we investigated the effect of TFP on CaM/Fas binding with molecular dynamics simulations. Conformation and binding free energy analyses were performed to examine the connections between the conformational changes of CaM by TFP and CaM/Fas binding affinity. Conformational characteristics of Fas by TFP were also examined for the further determining TFP effects on Fas recruiting FADD to form DISC. Binding free energy analyses showed that CaM antagonist, TFP inhibited CaM binding to Fas. The results are consistent with experimental results. The further conformational analyses showed that TFP significantly changed the CaM conformation, resulted in the increased Fas conformational fluctuations and the degree of correlation between motions of the residues in Fas, which provides structural insight for Fas further binding to FADD for DISC formation. Understanding the molecular mechanisms of CaM antagonist TFP in CaM/Fas binding for Fas-mediated DISC formation should provide important insight into the function of CaM antagonists in regulating Fas-mediated apoptosis.Keywords: CaM antagonist TFP; CaM/Fas binding; DISC; binding free energy, conformational analysis