title: Identification of the Calmodulin-Binding Domains of Fas Death Receptor.
abstract: The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas-mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209-239 (Fas-Pep1) and 251-288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD-CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling pathway.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, United States of America.
authors.forename: Bliss J
authors.initials: BJ
authors.lastname: Chang
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, United States of America.
authors.forename: Alexandra B
authors.initials: AB
authors.lastname: Samal
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, United States of America.
authors.forename: Jiri
authors.initials: J
authors.lastname: Vlach
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, United States of America.
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Fernandez
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, United States of America.
authors.forename: Dewey
authors.initials: D
authors.lastname: Brooke
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, United States of America.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Prevelige
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, United States of America.
authors.forename: Jamil S
authors.initials: JS
authors.lastname: Saad
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D018281:Cholangiocarcinoma / Q000378:metabolism / Q000473:pathology; D002942:Circular Dichroism; D053200:Fas-Associated Death Domain Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000032:analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D013816:Thermodynamics; D019014:fas Receptor / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D053200:Fas-Associated Death Domain Protein; D010455:Peptides; D011994:Recombinant Proteins; D019014:fas Receptor