Abstract 4754: Quantitative characterization of calmodulin and Fas death domain interactions
Authors: Romone Fancy, Tiara Napier, Gu Jing, Jay M. McDonald, Tong Zhou, Yuhua Song
Journal: Cancer Research (2012)

Abstract The Fas death receptor-activated death inducing signaling complex (DISC) regulates apoptosis in a variety of cells, including cholangiocarcinoma and other cancer cells. Qualitative immunoprecipitation experiments demonstrate that calmodulin (CaM) binds to Fas in a calcium-dependent manner and the CaM binding site in Fas has been localized in the Fas death domain (Fas DD) (J Biol Chem 279: 5661-5666, 2004). The interaction of CaM and Fas regulates DISC formation, and the CaM antagonist, trifluoperazine modulates the binding of Ca2+-bound CaM to Fas thus affecting Fas-mediated DISC formation (J Cell Biochem 103(3): 788-799, 2008). Fas mutations that cause an alteration of the structure and/or function of Fas have been detected in many cancers (J Pathol 193(2), 162-169, 2001; Cancer Res 61(4): 1382-5, 2001). The valine 254 to asparagine (V254N) mutation of Fas DD is analogous to the identified mutant allele of Fas in lpr-autoimmune mice that have a deficiency in Fas-mediated apoptosis (Nature 356(6367):314-317, 1992). This mutation also results in the decreased binding with CaM based on immunoprecipitation experiments (J Biol Chem 279: 5661-5666, 2004). Quantitative characterization of CaM and Fas interaction is important for designing optimized antagonists to modulate Fas-mediated DISC formation and thus apoptosis. In this study, we quantitatively characterize the interactions of CaM with wild type Fas and with the Fas V254N mutant using isothermal titration calorimetry (ITC). Recombinant CaM, Fas DD wild type (WT), and a FasV254N mutant were expressed in Escherichia coli, purified by ion affinity chromatography followed by size exclusion chromatography and dialyzed into the buffer for ITC experiments. ITC results illustrate an endothermic binding characteristic between CaM and WT Fas DD and an entropy-driven interaction between CaM and WT Fas DD with binding entropy of 46.7 ± 5.3 cal/mol/deg and a binding constant of 1.6 x105 ± 1.6 x104 M−1 at 37oC. The binding of CaM and Fas DD was significantly decreased by the V254N mutation of Fas DD, showing a significantly decreased binding entropy (33.2 ± 0.001cal/mol/deg) and a threefold decrease in binding affinity (5.01 x104 ± 3.4 x103 M−1) at 37oC. The significantly decreased binding affinity of CaM with Fas by the V254N mutation of Fas DD could result from the change of electrostatic interactions and van der Waals interactions between CaM and Fas DD and from the conformational change of Fas DD caused by the V254N mutation, which was demonstrated in our computational study (Biophys J. 95(12):5913-5921, 2008). Results from this study provide the thermodynamic basis for further identification of novel strategies capable of effectively regulating Fas-mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4754. doi:1538-7445.AM2012-4754