Analysis of the Open and Closed Conformations of the β Subunits in Thermophilic F1-ATPase by Solution NMR

F1-ATPase, composed of α, β, γ, δ, and ɛ subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the α3β3γ complex. We have investigated the α3β3γɛΔC (ɛΔC, truncated ɛ) complex from thermophilic Bacillus PS3 (TF1′, 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 15N-labeled TF1′ were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the β subunit revealed that the open form of the β subunit in the TF1′ complex is similar to that of the free β monomer. The open β subunit in the TF1′ complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF1α3β3γ. On the other hand, the closed form of the β subunit in the TF1′ complex was shown to be distinct from that of the nucleotide-bound β monomer. This is consistent with a previous report that the closed form of the TF1β monomer could be a catalytically activated state. The loop between the N-terminal β-barrel and the central domain is highly flexible in the TF1′ complex, in contrast to that in the α3β3 hexamer, suggesting that it is affected by the presence of the γ subunit in this area.