Active-Site Structure of the Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR

• Published in: Biophysical journal 2014-01, Vol.106 (2), p.390-398
• Main Authors: Kang, Su-Jin, Todokoro, Yasuto, Yumen, Ikuko, Shen, Bo, Iwasaki, Iku, Suzuki, Toshiharu, Miyagi, Atsushi, Yoshida, Masasuke, Fujiwara, Toshimichi, Akutsu, Hideo
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cadmium; Catalytic Domain; Cell Membrane - enzymology; Cell Membrane - metabolism; Correlation; Lipid Bilayers - metabolism; Magnetic Resonance Spectroscopy; Membranes; Models, Molecular; Molecular Sequence Data; Nuclear magnetic resonance; Oligomers; Protein Multimerization; Protein Structure, Quaternary; Protein Subunits - chemistry; Protein Subunits - metabolism; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - metabolism; Sails; Spectra; Temperature
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2013.12.005

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 13C-13C correlation spectra of TFoc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the Cαi+1-Cαi correlation spectrum of specifically 13C,15N-labeled TFoc rings. The Cδ 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 Cγ 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.