Identification and Preparation of a Novel Chemokine Receptor-Binding Domain in the Cytoplasmic Regulator FROUNT |
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Authors: | Akihiro Sonoda Sosuke Yoshinaga Kaori Yunoki Soichiro Ezaki Kotaro Yano Mitsuhiro Takeda Etsuko Toda Yuya Terashima Kouji Matsushima Hiroaki Terasawa |
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Affiliation: | 1.Department of Structural BioImaging, Faculty of Life Sciences,Kumamoto University,Kumamoto,Japan;2.Department of Molecular Preventive Medicine, Graduate School of Medicine,The University of Tokyo,Tokyo,Japan |
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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. |
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