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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Acknowledgements
We are grateful to Dr. Shogo Misumi for the MALDI/TOF MS measurements. This work was supported in part by the Targeted Proteins Research Program (TPRP) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (to S.Y., E.T., Y.T., K.M. and H.T.), by Practical Research for Innovative Cancer Control from the Japan Agency for Medical Research and Development (AMED) (to S.Y., E.T., Y.T., K.M. and H.T.), by Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT) from AMED (to S.Y., M.T., Y.T., K.M. and H.T.), by a Grant-in-Aid for Young Scientists (B) (JP19790064) from MEXT (to S.Y.), by the KUMAYAKU Alumni Research Fund (to S.Y.), by a Sasakawa Scientific Research Grant from the Japan Science Society (to K.Y.), by the Global COE Program-Cell Fate Regulation Research and Education Unit from MEXT (to K.Y.), by the Adaptable and Seamless Technology Transfer Program through target-driven R & D (A-STEP), Japan Science and Technology Agency (JST) (to H.T.) and by the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University (to H.T.).
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Sonoda, A., Yoshinaga, S., Yunoki, K. et al. Identification and Preparation of a Novel Chemokine Receptor-Binding Domain in the Cytoplasmic Regulator FROUNT. Mol Biotechnol 59, 141–150 (2017). https://doi.org/10.1007/s12033-017-0002-2
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DOI: https://doi.org/10.1007/s12033-017-0002-2