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Structural basis for the binding of the membrane‐proximal C‐terminal region of chemokine receptor CCR 2 with the cytosolic regulator FROUNT
Author(s) -
Esaki Kaori,
Yoshinaga Sosuke,
Tsuji Tatsuichiro,
Toda Etsuko,
Terashima Yuya,
Saitoh Takashi,
Kohda Daisuke,
Kohno Toshiyuki,
Osawa Masanori,
Ueda Takumi,
Shimada Ichio,
Matsushima Kouji,
Terasawa Hiroaki
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13096
Subject(s) - micelle , chemistry , chemokine receptor , g protein coupled receptor , biophysics , receptor , binding site , biochemistry , chemokine , microbiology and biotechnology , biology , aqueous solution
The membrane‐proximal C‐terminal region (Pro‐C) is important for the regulation of G‐protein‐coupled receptors ( GPCR s), but the binding of the Pro‐C region to a cytosolic regulator has not been structurally analyzed. The chemokine receptor CCR 2 is a member of the GPCR superfamily, and the Pro‐C region of CCR 2 binds to the cytosolic regulator FROUNT . Studying the interaction between CCR 2 Pro‐C and FROUNT at an atomic level provides a basis for understanding the signal transduction mechanism via GPCR s. NOE ‐based NMR experiments showed that, when bound to FROUNT , CCR 2 Pro‐C adopted a helical conformation, as well as when embedded in dodecylphosphocholine micelles. A comparison of two types of cross‐saturation‐based NMR experiments, applied to a three‐component mixture of Pro‐C, FROUNT and micelles or a two‐component mixture of Pro‐C and micelles, revealed that the hydrophobic binding surface on Pro‐C for FROUNT mostly overlapped with the binding site for micelles, suggesting competitive binding of Pro‐C between FROUNT and micelles. Leu316 was important for both FROUNT and micelle binding. Phe319 was newly identified to be crucial for FROUNT binding, by NMR and mutational analyses. The association and dissociation rates of CCR 2 Pro‐C for lipid bilayer biomembranes were faster than those for FROUNT . We previously reported that FROUNT binding to CCR 2 is detectable even in unstimulated cells and increases in response to chemokine stimulation. Taken together, these results support a model of CCR 2 equilibrium: chemokine binding changes the conformational equilibrium of CCR 2 toward the active state, and Pro‐C switches its binding partner from the membrane to FROUNT . Database Protein Data Bank submission codes of FROUNT‐bound form and DPC micelle‐bound form are PDB ID 2MLQ and PDB ID 2MLO , respectively. Biological Magnetic Resonance Bank submission codes of the samples of 800 μ m 13 C/ 15 N‐labeled CCR2 Pro‐C mixed with 8 μ m unlabeled FROUNT and the samples of 200 μ m 13 C/ 15 N‐labeled CCR2 Pro‐C mixed with 10 m m DPC‐d38 are BMRB ID 19829 and BMRB ID 19828, respectively. Structured digital abstractFROUNT binds to CCR2 Pro-C by surface plasmon resonance ( 1 , 2 ) FROUNT and CCR2 Pro-C bind by nuclear magnetic resonance ( View interaction )