Conformational Change of H+-ATPase β Monomer Revealed on Segmental Isotope Labeling NMR Spectroscopy

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Abstract
F1-ATPase has been shown to be a stepwise molecular motor. Its rotation mechanism has been explained by the interaction of the γ axis with the open and closed forms of the β subunit. Although NMR should be a powerful method for elucidating its mechanism, its molecular size (473 amino acid residues, 52 kDa) is a major obstacle. We have applied segmental labeling based on intein ligation to the β subunit, and succeeded in assigning 89% of the NH (402/451), 89% of the Cα (417/473), 83% of the Cβ (357/431), and 90% of the CO (425/473) signals of the β subunit monomer. The secondary structures predicted from the chemical shifts of the main chain atoms and the relative orientations determined from residual dipolar couplings indicated that the subunit β monomer takes on the open form in the absence of nucleotide. Furthermore, the chemical shift perturbation and the residual-dipolar-coupling changes induced by nucleotide binding show that conformational change from the open to the closed form takes place on nucleotide binding. The intrinsic conformational change of the β subunit monomer induced by nucleotide binding must be one of the essential driving forces for the rotation of F1-ATPase.