Stepwise Propagation of the ATP-induced Conformational Change of the F1-ATPase β Subunit Revealed by NMR

Hiromasa Yagi,Nobumoto Kajiwara,Tomoyuki Iwabuchi,Kenya Izumi,Masasuke Yoshida,Hideo Akutsu

doi:10.1074/jbc.m808212200

Abstract

The rotation of F1-ATPase (F1) is driven by the open/close bending motion of the beta subunit. The mechanism underlying the bending motion was investigated for the F1beta monomer from thermophilic Bacillus PS3 (TF1beta) in solution, using mutagenesis and NMR. The hydrogen bond networks involving the side chains of Lys-164 (numbering for TF1beta; 162 for mitochondrial F1beta 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 freebeta subunits in the crystal structures of mitochondrial F1. The role of each amino acid residue was examined by Ala substitution. beta(K164A) reduced the affinity constant for 5'-adenyl-beta,gamma-imidodiphosphate by 20-fold and abolished the conformational change associated with nucleotide binding and the ATPase activity of alpha3beta(K164A)3gamma.beta(T165A) and beta(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 beta(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 Lys-164 to Thr-165. Residual dipolar coupling analysis revealed that the closed conformation of the beta monomer was more closed than that in the crystal structure and was different for MgATP- and MgADP-bound beta subunits. Actually, MgATP induced a conformational change around Tyr-307 (311 for MF1beta), whereas MgADP did not. The significance of these findings is discussed in connection with the catalytic rotation of F1-ATPase.

Highlights

F0F1-ATP synthase catalyzes ATP synthesis coupled with proton flow across membranes driven by the electrochemical potential of a proton gradient [1,2,3,4]
It was demonstrated that the conformational change of the ␤ subunit from the open to the closed form upon nucleotide binding was similar to that found in mitochondrial F1 (MF1) crystal structures and was an intrinsic property of the ␤ subunit
For analysis of TF1, the crystal structure of MF1 was used as a structural model, because the crystal structure of TF1␤ in the ␣3␤3 hexamer is very similar to the open form of MF1␤ [16]

Summary

EXPERIMENTAL PROCEDURES

Construction of Mutated ␤ Subunit Genes and Preparation of Mutant Proteins—The expression plasmids of ␤(K164A), ␤(T165A), ␤(D311A), ␤(R333A), ␤(R191A), ␤(R191Q), ␤(R191E), and ␤(Y307P) were obtained from pUC118␤ by oligonucleotide-directed mutagenesis [21]. For preparation of the deuterated [2H-H, F, Y] proteins, cells were grown on a minimal medium with a mixture of amino acids. L-Phenylalanine, L-histidine, and L-tyrosine were replaced by L-[2,3,4,5,6-2H]phenylalanine, DL-[␣-2,4-2H]histidine, and L-[2,6-2H]tyrosine, respectively. [2H-H, F, Y] represents the deuteration of a protein by these amino acids. Glyphosate was added to the culture medium to inhibit aromatic amino acid biosynthesis. The details are given elsewhere [19]. All mutant proteins were purified according to previous reports [19, 22]

Construction of Expression Plasmids for Segmentally Labeled

RESULTS

Wild type

DISCUSSION

Yoshida and Hideo Akutsu