NMR analyses of the interaction between CCR5 and its ligand using functional reconstitution of CCR5 in lipid bilayers.
CC-chemokine receptor 5 (CCR5) belongs to the G protein-coupled receptor (GPCR) family and plays important roles in the inflammatory response. In addition, its ligands inhibit the HIV infection. Structural analyses of CCR5 have been hampered by its instability in the detergent-solubilized form. Here, CCR5 was reconstituted into high density lipoprotein (rHDL), which enabled CCR5 to maintain its functions for >24 h and to be suitable for structural analyses. By applying the methyl-directed transferred cross-saturation (TCS) method to the complex between rHDL-reconstituted CCR5 and its ligand MIP-1alpha, we demonstrated that valine 59 and valine 63 of MIP-1alpha are in close proximity to CCR5 in the complex. Furthermore, these results suggest that the protective influence on HIV-1 infection of a SNP of MIP-1alpha is due to its change of affinity for CCR5. This method will be useful for investigating the various and complex signaling mediated by GPCR, and will also provide structural information about the interactions of other GPCRs with lipids, ligands, G-proteins, and effector molecules.
Yoshiura
Chie
C
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
Kofuku
Yutaka
Y
Ueda
Takumi
T
Mase
Yoko
Y
Yokogawa
Mariko
M
Osawa
Masanori
M
Terashima
Yuya
Y
Matsushima
Kouji
K
Shimada
Ichio
I
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D054405:Chemokine CCL3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000378:metabolism; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions
D054405:Chemokine CCL3; D008024:Ligands; D008051:Lipid Bilayers; D008075:Lipoproteins, HDL; D008823:Micelles; D019713:Receptors, CCR5; D012996:Solutions; C411362:dodecyl maltopyranoside; D008320:Maltose