Active-site structure of the thermophilic Foc-subunit ring in membranes elucidated by solid-state NMR.
FoF1-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the Foc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TFoc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TFoc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TFoc oligomer was confirmed to be a ring indistinguishable from that expressed in E. coli on the basis of the H(+)-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D (13)C-(13)C correlation spectra of TFoc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C(α)i+1-C(α)i correlation spectrum of specifically (13)C,(15)N-labeled TFoc rings. The C(δ) chemical shift of Glu-56, which is essential for H(+) translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H(+)-locked conformation with Asn-23. The chemical shift of Asp-61 C(γ) of the E. coli c ring indicated an involvement of a water molecule in the H(+) locking, in contrast to the involvement of Asn-23 in the TFoc ring, suggesting two different means of proton storage in the c rings.
Department of Biophysics and Chemical Biology, Seoul National University, Seoul, Republic of Korea.
Su-Jin
SJ
Kang
Institute for Protein Research, Osaka University, Suita, Japan.
Yasuto
Y
Todokoro
Institute for Protein Research, Osaka University, Suita, Japan.
Ikuko
I
Yumen
Institute for Protein Research, Osaka University, Suita, Japan.
Bo
B
Shen
Institute for Protein Research, Osaka University, Suita, Japan.
Iku
I
Iwasaki
Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan; Department of Molecular Biosciences, Kyoto Sangyo University, Kyoto, Japan.
Toshiharu
T
Suzuki
Institute for Protein Research, Osaka University, Suita, Japan.
Atsushi
A
Miyagi
Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan; Department of Molecular Biosciences, Kyoto Sangyo University, Kyoto, Japan.
Masasuke
M
Yoshida
Institute for Protein Research, Osaka University, Suita, Japan.
Toshimichi
T
Fujiwara
Institute for Protein Research, Osaka University, Suita, Japan; Department of Biophysics and Chemical Biology, Seoul National University, Seoul, Republic of Korea. Electronic address: akutsu@protein.osaka-u.ac.jp.
Hideo
H
Akutsu
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
D008051:Lipid Bilayers; D021122:Protein Subunits; D006180:Proton-Translocating ATPases