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Tag: Fluorescence Polarization

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PLoS One. 2015 Nov 3;10(11):e0141692. doi: 10.1371/journal.pone.0141692. eCollection 2015.

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

Gaidos G1, Panaitiu AE1, Guo B1, Pellegrini M1, Mierke DF1.
Fig 6. Model of R4 peptide/calmodulin complex.

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.

http://www.ncbi.nlm.nih.gov/pubmed/26529318

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Biochemistry. 2014  Nov 4;53(43):6776-85. doi: 10.1021/bi500861x. Epub 2014 Oct 23

Protein Engineering of the N-terminus of NEMO: structure stabilization and rescue of IKKβ binding.

Abstract

NEMO is a scaffolding protein that, together with the catalytic subunits IKKα and IKKβ, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-ĸB pathway. Rational drug design targeting the IKK binding site on NEMO would benefit from structural insight, but to date the structure determination of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK binding domain NEMO(44-111) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled-coil at the N-terminus, C-terminus or both ends of NEMO(44-111) present high thermal stability and cooperative melting, and most importantly restore IKKß binding affinity. We examined the consequences on structural content and stability by circular dichoism and nuclear magnetic resonance and measured binding affinity of each construct for IKKβ(701-745) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method to engineer short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile the rescuing of the binding affinity implies that a pre-ordered IKK-binding region of NEMO is compatible with IKK binding and the conformational heterogeneity observed in NEMO(44-111) may be an artifact of the truncation.

http://www.ncbi.nlm.nih.gov/pubmed/25286246

C-terminal coiled-coil adaptor fused to NEMO
C-terminal coiled-coil adaptor fused to NEMO

 

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Engineering a soluble parathyroid hormone GPCR mimetic.

Proteins. 2013 Dec 27. doi: 10.1002/prot.24503. [Epub ahead of print]

Abstract

We designed and characterized a soluble mimic of the parathyroid hormone (PTH) receptor (PTH1R) that incorporates the N-terminus and third extracellular loop of PTH1R, important for ligand binding. The engineered receptor (PTH1R-NE3) was conceived to enable easy production and the use of standard biochemical and biophysical assays for the screening of competitive antagonists of PTH. We show that PTH1R-NE3 is folded, thermodynamically stable and selectively binds PTH. We also demonstrate the utility of our mimic by identifying a small molecule that competes with PTH in our PTH1R-NE3-based fluorescence polarization assay. Antagonists to PTH1R, a transmembrane protein belonging to the class B G-protein coupled receptor family, may provide new therapeutic options for calcium metabolism diseases like humoral hypercalcemia of malignancy.

prot24503-fig-0001