Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin

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.

An engineered construct of cFLIP provides insight into DED1 structure and interactions

“…We tried to map the ll Article Structure 30, 1-11, February 3, 2022 7 interaction site in 1 H, 15 N HSQC titration experiments in which the spectrum of 15 N-labeled DED1ch was recorded in the presence of increasing amounts of unlabeled, Ca 2+ -bound CaM. Addition of CaM induces a progressive loss in signal intensity of the DED1ch resonances, starting with CaM to DED1ch ratios as low at 0.1, consistent with the previously determined dissociation constant of 2 mM (Gaidos et al, 2015) (Figures 6 and S6). Perturbation of NMR resonances in binding experiments is caused both by direct interaction at the binding site and by secondary effects due to conformational rearrangements (which can be long range).…”

“…Perturbation of NMR resonances in binding experiments is caused both by direct interaction at the binding site and by secondary effects due to conformational rearrangements (which can be long range). In CaM's canonical binding mechanism, also confirmed by the CaM/cFLIP-H6a peptide complex structure (Gaidos et al, 2015), CaM's N-and C-terminal lobes completely wrap around a helical motif of the binding partner (here H6a). This binding mode implies that a large number of DED1ch resonances will be significantly perturbed (Yap et al, 2000), with CaM binding affecting both H6a as well as residues surrounding the helix and with widespread spectral changes caused by the conformational rearrangement that cFLIP DED1ch would need to undergo to present H6a appropriately for CaM binding.…”

“…We have previously shown that H6a represents the binding interface to calmodulin (Gaidos et al, 2015). H6a is also the site of the RxDL charged triad described as a hallmark feature of DEDs (Krueger et al, 2001;Li et al, 2006;Majkut et al, 2014;Park, 2007;Yang, 2008).…”

“…We previously showed that the interaction between wild-type cFLIP DED1 and calmodulin (CaM) is primarily mediated by residues in helix H6a of DED1 (Gaidos et al, 2015). Briefly, it was shown by domain swapping studies between DED1 and DED2 of cFLIP that H6a of DED1 is responsible for binding CaM.…”

“…Primers used for the constructs are listed in Table S1. The final chimeric cFLIP DED1ch construct was initially introduced into a modified pET30a plasmid expressing a GB1 fusion tag (courtesy of Pei Zhou, Duke University (Zhou and Wagner, 2010)), but subsequently the GB1 tag was excised in a single cloning step through the same site-directed mutagenesis protocol described above, leaving an uncleavable hexa-histidine tag at the N-terminus (Gaidos et al, 2015). Human calmodulin cDNA was purchased from OriGene (CALM2 gene, cat # SC125707) and amplified by PCR to be introduced into a pET16b plasmid by site-directed mutagenesis (Gaidos et al, 2015).…”