Analysis of the open and closed conformations of the beta subunits in thermophilic F1-ATPase by solution NMR.
F(1)-ATPase, composed of alpha, beta, gamma, delta, and epsilon subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the alpha(3)beta(3)gamma complex. We have investigated the alpha(3)beta(3)gamma epsilon(Delta C) (epsilon(Delta C), truncated epsilon) complex from thermophilic Bacillus PS3 (TF(1)', 360 kDa) in the solution state by using the combination of extensive deuteration, segmental-labeling, and CRINEPT (cross-correlated relaxation-enhanced polarization transfer) NMR. Well-resolved CRINEPT-HMQC (heteronuclear multiple-quantum correlation) spectra of partially (15)N-labeled TF(1)' were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the beta subunit revealed that the open form of the beta subunit in the TF(1)' complex is similar to that of the free beta monomer. The open beta subunit in the TF(1)' complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF(1)alpha(3)beta(3)gamma. On the other hand, the closed form of the beta subunit in the TF(1)' complex was shown to be distinct from that of the nucleotide-bound beta monomer. This is consistent with a previous report that the closed form of the TF(1)beta monomer could be a catalytically activated state. The loop between the N-terminal beta-barrel and the central domain is highly flexible in the TF(1)' complex, in contrast to that in the alpha(3)beta(3) hexamer, suggesting that it is affected by the presence of the gamma subunit in this area.
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita 565-0871, Japan.
Masumi
M
Kobayashi
Hideo
H
Akutsu
Toshiharu
T
Suzuki
Masasuke
M
Yoshida
Hiromasa
H
Yagi
D001407:Bacillus / Q000201:enzymology*; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry*; D002384:Catalysis; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D012996:Solutions
D021122:Protein Subunits; D012996:Solutions; D025243:Bacterial Proton-Translocating ATPases