title: Structural and biophysical characterization of the interactions between the death domain of Fas receptor and calmodulin.
abstract: The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (Kd) of ~2 μM and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Fernandez
authors.forename: Alexandra B
authors.initials: AB
authors.lastname: Samal
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Bedwell
authors.forename: Yabing
authors.initials: Y
authors.lastname: Chen
authors.forename: Jamil S
authors.initials: JS
authors.lastname: Saad
mesh_terms: D000818:Animals; D055592:Biophysical Phenomena; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002151:Calorimetry; D053200:Fas-Associated Death Domain Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D020349:Surface Plasmon Resonance; D013629:Tamoxifen / Q000494:pharmacology; D013816:Thermodynamics; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D053200:Fas-Associated Death Domain Protein; D011994:Recombinant Proteins; D013449:Sulfonamides; D013629:Tamoxifen; D014268:Trifluoperazine; C017967:W 7
keywords: Apoptosis; Calmodulin; Cholangiocarcinoma; Fas; NMR; Surface Plasmon Resonance (SPR)