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Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin.

Gaidos G, Panaitiu AE, Guo B, Pellegrini M, Mierke DF - PLoS ONE (2015)

Bottom Line: By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIPL.Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement.In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, Dartmouth College, Hanover, NH, United States of America.

ABSTRACT
Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIPL and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIPL. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIPL and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.

No MeSH data available.


Related in: MedlinePlus

The interaction with calmodulin is mediated by the C-terminal part of cFLIP DED1.(A) Schematic representation of the C-terminal swaps between DED1 and DED2. DED1 segments are illustrated in blue and DED2 segments in red. (B) Constructs illustrated in A were used as prey in a pull-down assay on calmodulin-conjugated resin. DED1 constructs containing DED2 segments lose the ability to bind calmodulin (lanes 1–3), while DED2 constructs containing DED1 segments show a gain in binding ability for calmodulin (lanes 4–6). (C) Synthetic R2, R3, R4 peptides corresponding to the DED1 regions shown in A were titrated into the pull-down assay to displace GST-DED1 from calmodulin-conjugated resin; R2 and R4 show inhibitory activity, while R3 is ineffective. Only pull-down fractions are shown for simplicity. (D) Homology model of DED1 highlights the position of the R2, R3, and R4 regions. Amino acid sequences for the three peptides are shown next to the model.
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pone.0141692.g003: The interaction with calmodulin is mediated by the C-terminal part of cFLIP DED1.(A) Schematic representation of the C-terminal swaps between DED1 and DED2. DED1 segments are illustrated in blue and DED2 segments in red. (B) Constructs illustrated in A were used as prey in a pull-down assay on calmodulin-conjugated resin. DED1 constructs containing DED2 segments lose the ability to bind calmodulin (lanes 1–3), while DED2 constructs containing DED1 segments show a gain in binding ability for calmodulin (lanes 4–6). (C) Synthetic R2, R3, R4 peptides corresponding to the DED1 regions shown in A were titrated into the pull-down assay to displace GST-DED1 from calmodulin-conjugated resin; R2 and R4 show inhibitory activity, while R3 is ineffective. Only pull-down fractions are shown for simplicity. (D) Homology model of DED1 highlights the position of the R2, R3, and R4 regions. Amino acid sequences for the three peptides are shown next to the model.

Mentions: Since to date there are no available structures for the DEDs of cFLIPL or its mammalian isoforms, we generated a homology model for cFLIP DED1 based on the structure of the DED1 of viral FLIP MC159 (PDB ID: 2BBR.1.A) using the SWISS-MODEL server [31, 32]. Inspection of this model reveals that the three regions identified through BLAST screening map in large extent to three putative helices in the C-terminal portion of DED1. These regions are highlighted on the homology model in Fig 3D. Based on their amphipathic helical projections, we targeted regions 2 and 4 of DED1 and designed DED chimeric proteins in which these regions in DED1 and DED2 were swapped. The corresponding R2–R4 regions on DED2 were delineated based on sequence alignment with DED1. The chimeric constructs are illustrated schematically in Fig 3A. Folding and stability of the hybrid constructs were confirmed by circular dichroism (S3 Fig). All constructs exhibit the predicted alpha helical character (S3A and S3E Fig) and are thermodynamically stable, as indicated by the elevated melting points (S3B–S3D and S3F–S3H Fig).


Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin.

Gaidos G, Panaitiu AE, Guo B, Pellegrini M, Mierke DF - PLoS ONE (2015)

The interaction with calmodulin is mediated by the C-terminal part of cFLIP DED1.(A) Schematic representation of the C-terminal swaps between DED1 and DED2. DED1 segments are illustrated in blue and DED2 segments in red. (B) Constructs illustrated in A were used as prey in a pull-down assay on calmodulin-conjugated resin. DED1 constructs containing DED2 segments lose the ability to bind calmodulin (lanes 1–3), while DED2 constructs containing DED1 segments show a gain in binding ability for calmodulin (lanes 4–6). (C) Synthetic R2, R3, R4 peptides corresponding to the DED1 regions shown in A were titrated into the pull-down assay to displace GST-DED1 from calmodulin-conjugated resin; R2 and R4 show inhibitory activity, while R3 is ineffective. Only pull-down fractions are shown for simplicity. (D) Homology model of DED1 highlights the position of the R2, R3, and R4 regions. Amino acid sequences for the three peptides are shown next to the model.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631386&req=5

pone.0141692.g003: The interaction with calmodulin is mediated by the C-terminal part of cFLIP DED1.(A) Schematic representation of the C-terminal swaps between DED1 and DED2. DED1 segments are illustrated in blue and DED2 segments in red. (B) Constructs illustrated in A were used as prey in a pull-down assay on calmodulin-conjugated resin. DED1 constructs containing DED2 segments lose the ability to bind calmodulin (lanes 1–3), while DED2 constructs containing DED1 segments show a gain in binding ability for calmodulin (lanes 4–6). (C) Synthetic R2, R3, R4 peptides corresponding to the DED1 regions shown in A were titrated into the pull-down assay to displace GST-DED1 from calmodulin-conjugated resin; R2 and R4 show inhibitory activity, while R3 is ineffective. Only pull-down fractions are shown for simplicity. (D) Homology model of DED1 highlights the position of the R2, R3, and R4 regions. Amino acid sequences for the three peptides are shown next to the model.
Mentions: Since to date there are no available structures for the DEDs of cFLIPL or its mammalian isoforms, we generated a homology model for cFLIP DED1 based on the structure of the DED1 of viral FLIP MC159 (PDB ID: 2BBR.1.A) using the SWISS-MODEL server [31, 32]. Inspection of this model reveals that the three regions identified through BLAST screening map in large extent to three putative helices in the C-terminal portion of DED1. These regions are highlighted on the homology model in Fig 3D. Based on their amphipathic helical projections, we targeted regions 2 and 4 of DED1 and designed DED chimeric proteins in which these regions in DED1 and DED2 were swapped. The corresponding R2–R4 regions on DED2 were delineated based on sequence alignment with DED1. The chimeric constructs are illustrated schematically in Fig 3A. Folding and stability of the hybrid constructs were confirmed by circular dichroism (S3 Fig). All constructs exhibit the predicted alpha helical character (S3A and S3E Fig) and are thermodynamically stable, as indicated by the elevated melting points (S3B–S3D and S3F–S3H Fig).

Bottom Line: By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIPL.Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement.In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, Dartmouth College, Hanover, NH, United States of America.

ABSTRACT
Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIPL and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIPL. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIPL and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.

No MeSH data available.


Related in: MedlinePlus