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Tag: protein engineering

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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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Reining in polyoma virus associated nephropathy: design and characterization of a template mimicking BK viral coat protein cellular binding.

Biochemistry. 2012 Oct 16;51(41):8092-9. doi: 10.1021/bi300639d. Epub 2012 Oct 2.

Abstract

The BK polyoma virus is a leading cause of chronic post kidney transplantation rejection. One target for therapeutic intervention is the initial association of the BK virus with the host cell. We hypothesize that the rate of BKV infection can be curbed by competitively preventing viral binding to cells. The X-ray structures of homologous viruses complexed with N-terminal glycoproteins suggest that the BC and HI loops of the viral coat are determinant for binding and thereby infection of the host cell. The large size of the viral coat precludes it from common biophysical and small molecule screening studies. Hence, we sought to develop a smaller protein template incorporating the identified binding loops of the BK viral coat in a manner that adequately mimics the binding characteristics of the BK virus coat protein to cells. Such a mimic may serve as a tool for the identification of inhibitors of BK viral progression. Herein, we report the design and characterization of a reduced-size and soluble template derived from a four-helix protein-TM1526 of Thermatoga maritima archaea bacteria-which maintains the topological display of the BC and HI loops as found in the viral coat protein, VP1, of BKV. We demonstrate that the GT1b and GD1b sialogangliosides, which bind to the VP1 of BKV, also associate with our BKV template. Employing a GFP-tagged template, we show host cell association that is dose dependent and that can be reduced by neuraminidase treatment. These data demonstrate that the BKV template mimics the host cell binding observed for the wild-type virus coat protein VP1.

http://www.ncbi.nlm.nih.gov/pubmed/23002929

NIHMS412129.html

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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