Vol.:(0123456789) 1 3 Biomolecular NMR Assignments https://doi.org/10.1007/s12104-018-9819-2 ARTICLE 1 H, 13 C and  15 N resonance assignments for a chemokine receptor- binding domain of FROUNT, a cytoplasmic regulator of chemotaxis Sosuke Yoshinaga 1  · Norihito Ishida 1  · Tatsuichiro Tsuji 1  · Akihiro Sonoda 1  · Kaori Yunoki 1  · Mitsuhiro Takeda 1  · Etsuko Toda 2  · Yuya Terashima 2  · Kouji Matsushima 2  · Hiroaki Terasawa 1 Received: 6 February 2018 / Accepted: 26 March 2018 © Springer Science+Business Media B.V., part of Springer Nature 2018 Abstract FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of infammatory immune cells. Therefore, FROUNT is a potential drug target for infammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previ- ously identifed the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/ P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain 1 H, 13 C, and 15 N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the 1 H– 15 N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors. Keywords Infammation · Cell migration · Chemokine receptor-binding domain · CCR2 · CCR5 · GPCR Biological context Infammation is an initial biological defensive mechanism against bacterial infection. Immune cells, such as mono- cytes and macrophages, sense the concentration gradients of chemokines and migrate to the infected site. This cell migra- tion is triggered by chemokines, which bind to G protein- coupled receptors (GPCRs) on the cell membrane. We identifed FROUNT as a cytoplasmic protein that binds to the C-terminal membrane proximal region (Pro-C) of the chemokine receptor, CCR2. The interaction between FROUNT and CCR2 regulates the migration of immune cells (Terashima et al. 2005). FROUNT was also found to bind CCR5, and thus it regulates CCR5-mediated cell migration (Toda et al. 2009). The FROUNT protein pro- duced by Escherichia coli naturally oligomerized (Esaki et al. 2011). A 16 amino acid region on the side of CCR2 Pro-C was identifed as the FROUNT binding region (Toda et al. 2014). The 16 amino acid peptide adopts an amphi- pathic helical conformation upon binding to FROUNT and interacts with FROUNT via its hydrophobic surface, as revealed by NMR (Esaki et al. 2014). The C-terminal region (residues 532–656) of FROUNT, named CRBD here, was identifed as the structural domain responsible for bind- ing to CCR2 Pro-C. However, the recombinant protein of CRBD (14.5 kDa) exhibited signifcant aggregation pro- pensity at a concentration of 100 µM (Sonoda et al. 2017). We screened 1,6-hexanediol as a solubilizing compound for the CRBD protein, using multiple terminal deletion mutants (Yunoki et al. 2018). We assigned the main-chain resonances of CRBD by deuteration and TROSY. However, the side- chain resonance assignment of CRBD was difcult due to its aggregation tendency. We also introduced L538E/P612S mutations to the CRBD protein, referred to as CRBD_LEPS, to mitigate its aggregation propensity and thereby improve the quality of obtainable NMR spectra, with no infuence * Hiroaki Terasawa terasawa@structbiol.com 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, Bunkyo-ku, Tokyo, Japan