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The rotation of F[sub1]-ATPase (F[sub1]) is driven by the open/close bending motion of the β subunit. The mechanism underlying the bending motion was investigated for the F[sub1]β monomer from thermophilic Bacillus PS3 (TF[sub1]β) in solution, using mutagenesis and NMR. The hydrogen bond networks involving the side chains of Lys-164 (numbering forTF[sub1]β 162 for mitochondrial F[sub1]β in parentheses), Thr-165(163), Arg-191(189), Asp-252(256), Asp-311(315), and Arg-333(337) in the catalytic region are significantly different for the ligand-bound and free βsubunits in the crystal structures of mitochondrial F[sub1]. The role of each amino acid residue was examined by Ala substitution. β(K164A) reduced the affinity constant for 5'-adenyl-β,γ-imidodiphosphate by 20-fold and abolished the conformational change associated with nucleotide binding and the ATPase activityof α[sub3]β(K164A)[sub3]γ ∙ β(T165A)and β(D252A)exhibited no effect on the binding affinity but abolished the conformational change and the ATPase activity. The chemical shift perturbation of backbone amide signals of the segmentally labeled β(mutant)s indicated stepwise propagation of the open/close conversion on ligand binding. The key action in the conversion is the switching of the hydrogen-bonding partner of Asp-252 from Lys164 to Thr-165. Residual dipolar coupling analysis revealed that the closed conformation of the β monomer was more closed than that in the crystal structure and was different for MgATPand MgADP-bound βsubunits. Actually, MgATP-induced a conformational change around Tyr-307 (311 for MF[sub1]β), whereas MgADP did not. The significance of these findings is discussed in connection with the catalytic rotation of F[sub1]-ATPase. [ABSTRACT FROM AUTHOR]

The ϵ subunit of bacterial and chloroplast FoF1-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound ϵ subunit from a thermophilic Bacillus PS3 at 1.9-Å resolution. The C-terminal two α-helices were folded into a hairpin, sitting on the β sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli e subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 ϵ subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the ϵ C- terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of FoF1-ATP synthase [ABSTRACT FROM AUTHOR]

The ϵ subunit of bacterial and chloroplast FoF1-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound ϵ subunit from a thermophilic Bacillus PS3 at 1.9-Å resolution. The C-terminal two α-helices were folded into a hairpin, sitting on the β sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli e subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 ϵ subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the ϵ C- terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of FoF1-ATP synthase [ABSTRACT FROM AUTHOR]