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 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.
Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.
Akihiro
A
Sonoda
Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.
Sosuke
S
Yoshinaga
Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.
Kaori
K
Yunoki
Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.
Soichiro
S
Ezaki
Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.
Kotaro
K
Yano
Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.
Mitsuhiro
M
Takeda
Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Etsuko
E
Toda
Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Yuya
Y
Terashima
Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Kouji
K
Matsushima
Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan. terasawa@structbiol.com.
Hiroaki
H
Terasawa
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000648:ultrastructure; D011485:Protein Binding; D019718:Receptors, CXCR4 / Q000737:chemistry* / Q000648:ultrastructure
C486264:Cxcr4 protein, rat; C503214:NUP85 protein, human; D028861:Nuclear Pore Complex Proteins; D019718:Receptors, CXCR4
Cell migration; Chemokine signaling; Construct optimization; GPCR; NMR; Structural domain