Identification and Preparation of a Novel Chemokine Receptor-Binding Domain in the Cytoplasmic Regulator FROUNT

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...

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Published in:	Molecular biotechnology Vol. 59; no. 4-5; pp. 141 - 150
Main Authors:	Sonoda, Akihiro, Yoshinaga, Sosuke, Yunoki, Kaori, Ezaki, Soichiro, Yano, Kotaro, Takeda, Mitsuhiro, Toda, Etsuko, Terashima, Yuya, Matsushima, Kouji, Terasawa, Hiroaki
Format:	Journal Article
Language:	English
Published:	New York Springer US 01-05-2017 Springer Nature B.V
Subjects:	Amino Acid Sequence Anti-inflammatory agents Binding Sites Biochemistry Biological Techniques Biotechnology Cell adhesion & migration Cell Biology Chemistry Chemistry and Materials Science Chemokines Chromatography Cloning, Molecular - methods Cytoplasm Escherichia coli - genetics Escherichia coli - metabolism Human Genetics NMR Nuclear magnetic resonance Nuclear Pore Complex Proteins - biosynthesis Nuclear Pore Complex Proteins - chemistry Nuclear Pore Complex Proteins - ultrastructure Original Paper Protein Binding Protein Science Proteins Receptors, CXCR4 - chemistry Receptors, CXCR4 - ultrastructure Signal transduction GPCR NMR Construct optimization Structural domain Chemokine signaling Cell migration
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1073-6085 1559-0305
DOI:	10.1007/s12033-017-0002-2