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Identification and Preparation of a Novel Chemokine Receptor-Binding Domain in the Cytoplasmic Regulator FROUNT.
- Source :
-
Molecular biotechnology [Mol Biotechnol] 2017 May; Vol. 59 (4-5), pp. 141-150. - Publication Year :
- 2017
-
Abstract
- FROUNT is a cytoplasmic protein that binds to the membrane-proximal C-terminal regions (Pro-Cs) of chemokine receptors, CCR2 and CCR5. The FROUNT-chemokine receptor interactions play a pivotal role in the migration of inflammatory immune cells, indicating the potential of FROUNT as a drug target for inflammatory diseases. To provide the foundation for drug development, structural information of the Pro-C binding region of FROUNT is desired. Here, we defined the novel structural domain (FNT-CB), which mediates the interaction with the chemokine receptors. A recombinant GST-tag-fused FNT-CB protein expression system was constructed. The protein was purified by affinity chromatography and then subjected to in-gel protease digestion of the GST-tag. The released FNT-CB was further purified by anion-exchange and size-exclusion chromatography. Purified FNT-CB adopts a helical structure, as indicated by CD. NMR line-broadening indicated that weak aggregation occurred at sub-millimolar concentrations, but the line-broadening was mitigated by using a deuterated sample in concert with transverse relaxation-optimized spectroscopy. The specific binding of FNT-CB to CCR2 Pro-C was confirmed by the fluorescence-based assay. The improved NMR spectral quality and the retained functional activity of FNT-CB support the feasibility of further structural and functional studies targeted at the anti-inflammatory drug development.
- Subjects :
- Amino Acid Sequence
Binding Sites
Cloning, Molecular methods
Escherichia coli genetics
Nuclear Pore Complex Proteins ultrastructure
Protein Binding
Receptors, CXCR4 ultrastructure
Escherichia coli metabolism
Nuclear Pore Complex Proteins biosynthesis
Nuclear Pore Complex Proteins chemistry
Receptors, CXCR4 chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1559-0305
- Volume :
- 59
- Issue :
- 4-5
- Database :
- MEDLINE
- Journal :
- Molecular biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 28342149
- Full Text :
- https://doi.org/10.1007/s12033-017-0002-2